R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 ( 1 ) : 5 9 - 7 1 , j a n - m a r , 1 9 9 2
A R T IG O D E R E V ISÃ O
M O L E C U L A R B IO L O G IC A L T E C H N IQ U E S F O R T H E
D IA G N O S IS O F IN F E C T IO U S D IS E A S E S
G eraldo B rasileiro Filho and Sérgio D an ilo Junho Pena
In fectio u s ag en ts h av e trad itio n ally been d etec ted in c lin ica l sam p les by m icro sco p ic identification, by isolation after culturing o r by d etectio n o f a specific h o st im m une response31- M icroscopy is useful in identifying bacteria, fungi and p arasites, b ut is generally n o t able to recognize viruses. A lth o u g h considered the m ost sensitive diagnostic m ethod, culture cannot b e used routinely in all cases, especially in viral diseases. The p ro g ress im m unology has achieved in recent years has m ade p o ssib le the detection o f m any m icro org an ism s th ro u g h im m unocytochem istry and by differen t serologic reactions using EL ISA and radioim m unoassay. H ow ever, som e infections cannot be identified du e to low am ount o r absence o f relevant antigens o r antibodies.
In th e last few y ears m o lecu lar biology experienced a trem endous advance and provided pow erful tools to diagnose many infectious diseases. T o d ay , D eso x y rib o se N ucleic A cid (D N A ) and R ib o se N u c le ic A c id (R N A ) te c h n o lo g y is becom ing increasingly used as effective options for th e d etection o f infectious agents. All m icrobes have th eir ow n in dividuality expressed at the genetic level in th eir sequences o f nucleic acids. T h e p in p o in tin g o f these specific sequences by the pro ced u res described below perm its accurate, sensitive and effective diagnosis o f a large num ber o f infectious diseases.
F U N D A M E N T A L S O F M O L E C U L A R H Y B R ID IZ A T IO N
T h e r a tio n a le f o r d ia g n o s tic m o le c u la r biolo g ical techiniques is based on nucleic acid
T r a b a l h o d o s D e p a r ta m e n to s d e A n a to m ia P a to ló g ic a e M e d ic in a L e g a l d a F a c u ld a d e d e M e d ic in a e d e B io q u ím ic a e Im u n o lo g ia , In s titu to d e C iê n c ia s B io ló g ic a s d a U n iv e rs id a d e F e d e r a l d e M in a s G e r a is , B e lo H o r iz o n te , M .G .
S u p p o rte d b y C N P q
Adress f o r correspondence: P r o f . G e r a ld o B ra s ile iro F ilh o , D e p t o . d e A n a to m ia P a to ló g ic a e M e d ic in a L e g a l/F M /U F M G . C a ix a P o s ta l 3 4 0 , 3 0 1 3 0 - 9 7 0 , B e lo H o r iz o n te -M G . R e c e b id o p a r a p u b lic a ç ã o e m 0 2 /0 2 /9 1 .
structure. D N A is chem ically m o n o to n o u s and structurally very sim ple. Its p rim ary stru ctu re is m ade by long chains o f o nly the fo u r nucleotides o f adenosine (A ), cytosine (C ), guan id in e (G) and thym idine (T), linked by p h o sp h o d iester bonds. Secondary strucutre is form ed by a d ouble helix stabilized by hydrogen bonds betw en adenine and thym idine (tw o bonds) and quanine an d cytosine (tree bonds). D N A is structurally v ery stable, b u t the tw o chains can b e separated by chem icals o r by heat, a process called denaturation is irrev ersib le, th e tw o D N A strands rejo in as soon as th e d enaturing agent is removed. T his renaturation is b oth fast and specific. T he speed com es fro m the p o sitiv e co operativity in the form ation o f m ultiple hyd ro g en bonds w hich give enorm ous stability to the doubel helix. T he specificity derives from the u n iq u e p airin g o f A w ith T and G w ith C. F o r the sam e reason, only exactly com plem entary sequences form stabel duplexes. Since the system has no m em ory, a single D N A strand can associate w ith th e stran d fro m w h ich it cam e apart o r w ith any o th e r stran d h av in g a com plem entary sequence. T h e latter is generally called hybridization. T he ability o f D N A to h y bridise w ith g reat specificity to k n ow n com plem entary s e q u e n c e s h a s d is c lo s e d n e w a n d im p o rta n t perspectives fo r m edical diagnosis. D N A probes, w ich are being used w ith increasing frequency w o r ld w id e , h a v e m a d e p o s s i b le th e r a p id identification o f m any m icro-organism s previously detectable o nly by tedious and tim e-consum ing m ethods. M o re recently, an o th er technique, the Polym erase Chain R eactions (P C R ), has had profound im pact o n m edical d iagnosis, by apm plifying in v i t r o specific nucleic acids sequences o f different infectious agents.
D N A PRO BES
A D N A probe is a know n D N A sequence obtained by m olecular coining (polynucleotides) o r by chemical s y n th e s is ( o l i g o n u c l e o t i d e s ) . T h e p r o b e is com plem entary to a D N A o r R N A sequence o f
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n iq u e s f o r th e d i a g n o s i s o f i n f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
in te r e s t (ta r g e t s e q u e n c e ) an d h a s a la b e l th a t m a k e s p o s s i b l e it s s e l e c t i v e id e n tific a t io n . W h e n th e ta rg e t is in it s n a t iv e p la c e ( in t is s u e s o f a h is t o lo g ic s e c t io n , in c e l l s o f a s m e a r o r in a c h r o m o s o m a l
p r e p a r a t i o n ; , t h e m e t h o d i s c a l l e d i n s i t u h ib r id iz a t ic i f t h e D N A is s o lu b iliz e d a n d b o u n to f i l t e r s o f i / t r o c e l u l o s e o r n y l o n p r i o r t o h ib r id iz a f' '? t is c a lle d a b lo t o r filte r h ib r id iza tio n . In d i ‘: ,r , tic p r o c e d u r e s , D N A p r o b e s b e h a v e m u c h 1 t ' b o d ie s , in th e s e n s e th a t th e y b in d a s p e c if ic ti " f a n d ca rry a la b e l. H o w e v e r , th e u s e o f i r o b e s h a s m a n y a d v a n t a g e s o v e r im ir m r '-d la g n o s is . F ir s t, D N A i s m u c h m o r e s ta b e l th a n t h e n jo r ity o f p r o te in s . S e c o n d , h y b r id iz a tio n w i t h D M A p r o b e s is in d e p e n d e n t o f g e n e t ic
e x p r s^ io n in th e s p e c im e n u n d e r stu d y . In th is
w a y , o n e c a n d ia g n o s e a v ir a l in fe c tio n b y fin d in g
th e v ir a l g e n o m e it s e l f in ste a d o f s e r a c h in g fo r
p r o t e in s th a t r e p r e s e n t p r o d u c ts o f v ir a l g e n e e x p r e s s io n . T h u s , it is p o s s ib le to d e te c t a v ir u s e v e n w h e n it is in a la te n t in f e c t io n , in th e fo r m o f a p r o v ir u s , a s o c c u r s w it h th e r e tr o v ir u s H I V 1.
P r o b e s ty p e s
T h e r e a re D N A a n d R N A p r o b e s . T h e fo rm er a r e u s e d m u c h m o r e f r e q u e n tly b e c a u s e o f th eir
g r e a te r s t a b ilit y . D N A p r o b e s a re o b ta in e d b y t e c h n iq u e s o f g e n e t ic e n g in e e r r in g a n d ca n b e o r ig in a te d fr o m th e g e n o m e o f an o r g a n ism (g e n o m ic p r o b e s ) o r m a d e th r o u g h th e r e v e r s e tra n scr ip tio n o f R N A ( c o m p le m e n t a r y o r c D N A p r o b e s ) . O li g o n u c le o t id e p r o b e s ( 2 0 - 1 0 0 n u c le o t id e s lo n g ) a re m a d e b y c h e m ic a l s y s t h e s is in s o li d p h a se . T h e t e c h n ic a l a c h ie v e m e n t s in th is la tte r f ie l h a v e b e e n g r e a t a n d t h e s e p r o b e s a re p r o g r e s s iv e ly r e p la c in g c lo n e d p r o b e s - f o r m a n y h ib r id iz a t io n s y s t e m s th e y a r e p r e fe r r e d .
L a b e llin g
T h e m o s t c o m m o ly u s e d t y p e o f p r o b e la b e llin g is th e in c o r p o r a tio n o f r a d io c tiv e is o t o p e s , e s p e c ia lly P h o s p h o r u s ( P ) 32, w h ic h p r o v id e s h ig h s e n s itiv ity . H o w e v e r , th e d r a w b a c k s o f h ig h c o s t s , sh o r t h a lf-l i v e , h e a lf-lth r is k s a n d t h e lf-lo g is t ic s o f h a n d lf-lin g r a d io c t iv e m a te r ia ls in a c lin ic a l la b o r a to r y h a v e
m o tiv a t e d th e d e v e lo p m e n t o f s y s t e m s o s n o n i s o t o p i c l a b e l l i n g ( c o l d p r o b e s ) . M a n y n o n r a d io a c tiv e c h e m ic a ls , m a in ly b io t in , h a v e b e e n lin k e d s u c e s s f u ll y t o p r o b e s an d s h o w e d t o b e
v a lu a b le in v a r io u s h ib r id iz a t io n p r o c e d u r e s . T h e u s e o f p r o b e s c o n j u g a t e d d ir e c t ly t o a lk a lin e p h o sp h a ta se r e p r e s e n ts a n o th e r a d v a n c e in th is area, s in c e th ey ca n b e a ssa y e d e a s ily in a co n v e n tio n a l la b o ra to ry s e ttin g an d th e r e s u lts o b ta in e d w it h th e m are a d e q u a te . T h e m a in d is a d v a n t a g e o f t h e s e “ c o ld p r o b e s ” is th e ir lo w e r s e n s i t iv i t y , a lth o u g h s o m e in v e s t ig a t o r s h a v e c la im e d to a c h ie v e r e s u lts c o m p a r a b le to r a d io a c tiv e p r o b e s 6 57.
H y b r id iz a tio n
T h e d ia g n o s is o f in f e c t io u s d is e a s e s in c lin ic a l s a m p le s ca n b e d o n e b y in s i t u o r f ilt e r h y b r id iz a t io n ( b lo t ) . F o r in s i t u h y b r id iz a t io n ( I S H ) o n e u s e s h is t o lo g ic a l s e c t io n s f r o m s a m p le s o b ta in e d in s u r g e r y ( b io p s y ) o r a fte r d ea th ( a u t o p s y ) , o r e v e n c e llu la r sm ea rs su c h a s b lo o d c e lls , m u c o sa l sc ra p in g s ( c e r v ic a l, b r o n q u ia l e t c .) o r s e d im e n t s o f o r g a n ic f lu id s (u r in e , a s c it ic f lu id e t c .) . I n ia t ia lly , th e p r e p a r a tio n s a re trea te d w it h p r o t e a s e s in o r d e r to p e r m it p r o b e a c c e s s , to e x p o s e th e ta rg e t s e q u e n c e an d to lo w e r th e n o n - s p e c if ic a d s o r p tio n o f th e p r o b e . S u b s e q u e n tly , th e s a m p le s a r e d en a tu r e d b y h ea t, h y b r id is e d an d d e v e lo p e d in m ic r o s c o p e s lid e s . T h e in fo r m a tio n g a in e d is p r e c io u s , s in c e th e t e c h n i q u e d e t e c t s i n f e c t i o u s a g e n t s in t h e ir to p o g r a p h ic a l s it e w it h in th e str u c tu r e o f th e t is s u e .
M o r e o v e r , IS H a llo w s : 1. th e a n a ly s is o f sm a ll s a m p le s , w it h o n ly a f e w c e lls ; 2 . th e id e n tific a t io n o f a m ic r o -o r g a n is m e v e n w h e n o n ly a m in o r it y o f c e lls is in fe c te d ; 3 . to k n o w i f th e in f e c t io u s a g e n t is in in s id e a le s io n ( e . g . a n e o p la s ia ) o r i f it is in n o r m a l a d ja c en t tis s u e s ; 4 . r e t r o s p e c tiv e s t u d ie s , s in c e f ix e d m a te ria l in p a r a ffin b lo c k s c a n b e
a n a ly s e d w it h s u c c e s s ; 5 . s in c e it is a b le to d e te c t m R N A , it c a n in fo r m a b o u t g e n e t ic e x p r e s s io n o f th e m ic r o -o r g a n is m .
F ilte r h y b r id iz a tio n a lw a y s d e p e n d s o n p r e v io u s s o lu b iliz a tio n o f th e n u c le ic a c id s p r e s e n t in th e s a m p le s to b e a n a ly s e d . F o r d o t b lo t s , n u c le ic a c id s are a d so r b e d d ir e c tly to filte r . In S o u th e r n b lo ts D N A is in itia lly c le a v e d b y re stric tio n e n d o n u c le a s e s an d th e r e s u ltin g f r a g m e n ts a r e s e p a r e te d a c c o r d in g to th e ir s iz e b y e le c tr o p h o r e s is in a g a r o s e g e l s , t r a n s fe r r e d t o t h e f il t e r a n d h y b r id i s e d w i t h
a p pr op ria ted p r o b e s. F o r f ilte r h y b r id iz a tio n , s o lu b e D N A is m o r e p r o p e r ly o b ta in e d f r o m fr e sh s a m p le s , a lth o u g h th e re a re te c n iq u e s th at p e r m it n u c le ic a c id s o lu b iliz a tio n a n d is o la t io n f r o m h is t o lo g i a l s e c t io n s
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l t e c h i n i q u e s f o r th e d i a g n o s i s o f in f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
(h is to b lo ts ) . T h e m ain a d v a n ta g e s o f f ilte r h y b rid izatio n are h igh sensitivity and specificity, besides the fact that o f sam ples can be analysed sim ultaneosly.
D evelopm ent
F o r radioactive probes the developm ent consists on au toradiography o f filters o r m icroscopic slides. F o r n o n -radioactive probes, the hybridization result appears as a fluorescent o r colored signals, visible at the m icroscope (ISH ) o r the naked eye (blots).
In h y b rid izatio n w ith biotinylated probes the signal depends on th e appearence o f a chrom ogenic substance form ed by an enzym e o ver a substrate. T h e enzym e (com m only peroxidase or alkaline phosphatase) is conjugated to streptavidin o r avidin, w h ic h in tu r n h as g re a t a f in ity to b io tin . H y b rid izatio n having occured, a com plex biotin- avidin-enzym e-substrate is form ed at sites o f probe h y b rid izatio n w ith the target and results in a colored precipitate.
F ig u re 1 show s the basic aspects o f m olecular h y b rid izatio n w ith D N A probes.
P O L Y M E R A S E C H A IN R E A C T IO N
In the sh o rt h isto ry o f m olecular biology, only a few events have had so m uch im pact than the d iscovery in 1985 o f the technique o f Polym erase C hain R eaction (P C R ), a m ethod that perm its the am plification o f specific segm ents o f nucleic acids. P C R is based o n repetitive cycles o f in v itr o synthesis o f D N A segm ent, using a D N A polym erase in presence o f nucleotides and co-factors. In the fis rt step th e n ativ e d o u b le-stran d ed D N A is d enatured by heat. In the second step two D N A prim ers are anneled to com plem entary sequences on o p p o site strands o f the tem plate D N A and thus fla n k th e re g io n o f in te re s t. F in a lly , D N A po ly m erase synthesises a com plem entary strand o f D N A through thé extension o f each annealed p rim er. A n essential feature in PC R is that the products o f a m p lifica tio n act as tem plates fo r subsequent synthesis. In the n ex t cycle, the two double-stranded D N A segm ents are again denatured to prim ers and copied (F ig u re 2). B y repeating these cycles one o btains an exponential D N A synthesis, since in each cycle th e n um ber o f segm ents o f interest is d o u b le d ( h e n c e th e n a m e c h a in r e a c tio n ) .
Theoretically, after 20 cycles o f am plification 220 (1 m illion) copies are obtained. So, a single copy o f a D N A sequence (e.g. on e gene o r one v iru s p er cell) can b e reco g n ized by th e e x tra o rd in a ry sensitivity o f this technique.
W ith the advent o f p rogram m able therm al cyclers and the discovery o f T aq Polym erase, a therm ostable D N A polym erase isolated from a therm ophilic bacteria, P C R tecnique is no w a partially autom ated pro ced u re th at p erm its the analysis o f m any sam ples at the sam e tim e.
T he am plifyed D N A , intact o r after digestion by restrictio n enzym es, can b e id en tified and characterized by d ot blots, by Southern blots o r, sim ply, by electrophoretic m igration in agarose or polyacrylam ide gel.
T h u s , P C R e m e r g e s as a s im p le a n d program m able procedure, having properties o f great specificity (adjustable by selection o f th e prim ers and tem p eratu re o f a n n ealin g ) an d en o rm o u s sensitivity (controled by the n u m b er o f cycles, usually betw en 20 and 40). It is n o t surprising, th erefo re, that P C R is ra p id ly b eco m in g the technique o f choice fo r d iag n o sin g infectio u s diseases by am plifying sh o rt genom ic segm ents o f viruses, bacteria, fungi o r protozoa, even in presence o f a large excess o f host D N A . H ow ever, the use o f PC R for m edical diagnosis should b e exercised w ith extrem e care, since its best advantage is also its m ajor draw back: due to its enorm ous sensitivity, contam ination o f the reaction w ith even a single m olecule o f the p roduct u n d er study can p ro d u ce a false-positive result.
All these tools have revolutionised the diagnosis o f infectious diseases. H ow ever, this technology is still largely a privilege o f research laboratories and usually confined to the academ ic environm ent. Its w idespread application for m edical p ractiv e w ill require years o r decades. Y et, new sim plified approaches to som e o f these techniques are being developed and p rogressively applied to solve daily m edical problem s.
D IA G N O SIS O F IN F E C T IO U S D ISEA SES
F o r space lim itations, a com plete review o f all infectious agents cannot b e d one in this article. O nly those m ore im portant v iru ses, bacteria and parasites w ill b e discussed.
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n i q u e s f o r t h e d i a g n o s i s o f i n f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1, j a n - m a r , 1 9 9 2 .
V IR A L D ISEA SES H E PA TIT IS
T h e techniques o f m olecular hybridization w ith D N A p ro b e s an d P C R have becom e valuable m e th o d s o f a n a ly s i s a n d in v e s t i g a t i o n in m icro b io lo g y . G reater p ro g ress in this area has been observed in th e study o f v iral diseases.
H epatitis B
T he diagnosis o f hepatitis B is rou tin ely m ade by the detection in serum o f specific antigens o r antibodies. H ow ever, these serologic m arkers are inconstant and th eir absence does n o t ru le out
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n i q u e s f o r th e d i a g n o s i s o f in f e c t i o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
infection by the v iru s (H BV) n o r indicates absence o f infectivity. D irect virus detection is difficult by lack o f a sim ple an d practical system o f cell culture.
In th e p ast few years there has been a grow ing in terest in v iral D N A detection in serum o r in liver using S outhern o r d ot blot, w hich can detect HBV D N A even w h en th eir serologic m arkers are n ot found5. By d o t blo t w ith radioctive probes, one can detect low quantities o f v iru s in blood, o f the order
o f 104 particles per sam ple. H ow ever, this procedure is no t able to detect sm aller am ounts o f v iru s still s u f f ic ie n t f o r t r a n s m ittin g th e d is e a s e . In chimpanzees, for instance, 102 o r m ore viral particles can transm it the infection.
M o r e r e c e n t l y , P C R te c h n o l o g y h a s revolutionised this area. U sin g d ifferen t p rim er sets and oligonucleotide p ro b es, it is n o w p o ssilb le to detect HBV w hen present in levels o f 101 - l 02
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n i q u e s f o r th e d i a g n o s i s o f i n f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
p a rtic le s/m l37 64. T herefore, P C R is the m ost sensitive m ethod n o t only fo r diagnosis b u t also for th e screening o f sam ples in blood transfusion centers.
H ep atitis C
T h e great m ajority o f post-transfusion hepatitis w o rld w id e is o f non-A , non-B type (N A N B H ), w hich is caused by the hepatitis C virus (H C V ), an R N A v ir u s o n ly re c e n tly c h a r a c te r is e d by re co m b in an t D N A tech n o lo g y 13.
C u rren t diagnosis o f hepatitis C and screening o f b lood d o nors is based on the detection o f a serum antibody directed to a recom binant protein coded by the v iru s (C 100-3)43. A lthough serelogic tests w ith an ti-C 100-3 have represented a substantial advance in H C V detection, there are som eproblem s. F isrt, n o t every b lo o d sam ple positive for anti- C100-3 has a real risk o f transm itting the disease23 67. Second, the test can give false-negative resu lts, as anti-C 100-3 is directed against the reg u lato ry and n o t to structural proteins o f the viral envelope. It is also negative w hen assayed before seroconversion, w hich occurs around 22 weeks after infection or 15 w eeks after th e onset o f clinical signs o f hepatitis; m oreover, in about 40 % o f acute N A N B H patients no antibody ever develops1.
T h e sequencing o f th e viral genom e has m ade p o ssib le th e d esig n o f P C R system s to analyse clinical sam ples fo r diagnosis in patients w ith liver disease and fo r screening o f blood donors23. D ue to its g reat sensitivity, P C R is able to identify very sm all am ounts o f the v irus, even in the absence o f its seru m m ark e rs. C u rre n tly , m any research lab o rato ries w o rld w id e are investing m uch in PC R technology fo r detection o f H C V , em phasizing out its effectiveness fo r diagnosis, epidem iological studies and selection o f b lood donors20 23.
O th er hepatitis
D N A probes o r P C R m ethods are also available fo r diagnosis o f hepatitis A 35, D 1671 and E52.
R o tavirus
H um an ro tav iru s is the m ost prevalent cause o f acute diarrh ea in children up to tw o years old. In practiv e, the diagnosis o f rotavirus infection is confirm ed by detectio n o f viral antigens by ELISA tests. It also can b e m ade by visualization o f viral p articles by electron m icroscopy (E M ) o r by R N A
analysis by p o ly acrylam ide gel electro p h o resis (P A G E). H ow ever, E M and P A G E are available only in research centers.
In la s t d e c a d e , m o le c u la r h y b r id iz a tio n technology started to be used and proved useful for detection o f ro tav iru ses A and B 17 19. U sing O ligonucleotide probes com plem entary to conserved regions o f the viral genom e, d ifferen t viral types could b e detected2. W hen cloned p ro b es derived fro m specific segm ents w e re em ployed, m any distinct serotypes could de identified70.
H um an papillom avirus
H um an papillom avirus (H PV ) has n otorius tropism for the squam ous ep ithelia o f skin and m ucous m em branes, w h ere it is associated w ith proliferative lesions o f v arying m alignant p o ten tial. T he m ost frequent and im portant lesions are located in genital system . T here, am ong m ore than 60 H PV types now know n, som e (m ainly types 6 and 11) have been im plicated in genesis o f benign o r low - grade lesions, w hereas others (notably types 16 and 18) have been associated p referentially w ith high- grade lesions or m alignant neoplasm s41.
A ccurate detection and p recise typing o f H PV is really relevant for b etter understanding o f the n atu ra l h is to ry , ep id em io lo g y an d o n co g en ic potential o f H PV . C onventional m ethods o f H PV detection usually have low sensitivity and n one can discrim inate viral types. F o r these reasons, am ong all v iral diseases, H PV infection is th e one th at has derived the to date largest b enefit fro m m olecular biology techniques. Besides d etecting the virus w ith g reat sen sitiv ity , m o lecu lar h y b rid izatio n techniques are unique in id entifying H P V types safely. A s stated above, this aspect is crucially relevant in H P V infection.
A ll m olecular biology procedures described previously have been used, largely w ith success, fo r the diagnosis o fH P V infection in genital lesions. T he sam ples can be o b tained by biopsy o r by cervico-vaginal scrapes. D o t blo ttin g is the sim plest and m ost practical m ethod, since it is easy to p erfo rm and m ultiple sam ples can b e tested at each tim e68. Its sensitivity, how ever, is low er than that o f oth er procedures. Southern b lo ttin g is very sensitive and considered the “ g old stan d ard ” for diagnosis o fH P V infection. O ther advantage o f this m ethod is the p ossibility o f studying H P V physical
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l l e c h i n i q u e s f o r th e d i a g n o s i s o f in f e c t i o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
state in cells40, since in the m ajority o f m alignant lesions th e v iral D N A is integrated in the host genom e. Y et, due to its laborious handling, Southern blo ttin g is p erform ed o nly in research centers. W ith the sim plification o f the m ethodology due to the av ailab ility o f n o n -rad io activ e probes, HIS in cerv ical tissu es can b e n o w ap p lied in m any laboratories. In o u r experience25, H IS has show n to b e useful fo r detecting and typing H PV in sam ples rou tin ely received fo r histopathological analysis. Its sensitivity is low er than that o f Southern b lo ttin g , b u t it has som e advantages o v er the last m ethod: 1. as in dividual cells are analysed, it can recognise the v iru s w hen only a few cells are infected; 2. perm its in fo rm atio n i f the v iru s is w ithin a lesion or if it is found in n orm al adjacent tissues.
T h e P C R technique is now being used w ith increasing frequency fo r H P V detection. W hen perfo rm ed w ith th e essential and desired care, P C R h a s s h o w n to b e v e r y e f f e c ti v e in H P V i d e n t i f i c a t i o n 42 62. U s in g p a ir s o f p r im e r s h o m ologous to a conserved region in m any H PV types, it w as p ossible to am plify sequence o f differen t types o f H P V 57 62. In p ositive cases, virus typing can be achieved by subsequent hybridization w ith s p e c if ic p r o b e s . T h is t e c h n o lo g ic a l im provem ent has g reat im portance, since it reduces tim e and reagents necessary to testing several sam ples.
A lth o u g h fresh sp ecim en s are p re fe rre d , m o lecular h y b rid izatio n tests can be done in old sam ples fixed for m onths o r years and stored in paraffin blocks. This is very valuable to retrospective studies, w hich are o f enorm ous im portance for the b etter u n derstanding o f H PV infection.
C ytom egalovirus
C ytom egalovirus (C M V ) is a m ajor cause o f congenital infection in hum ans, it has been estim ated that 60-80% o f in dividuals in developed countries and v irtu ally 100% o f those living in developing countries w ill b e infected w ith it d uring their lif e tim e 26. I n m o s t p e o p le th e in fe c tio n is asym ptom atic, b u t w hen th e im m unologial defense dro p s fo r any reason (im m unosuppressive drugs in allo g raft recipients, pacients w ith cancer AIDS etc .) it m ay becom e serious and can even be lethal. C M V can cause pneum onitis, hepatitis, colitis, infeccious m ononucleosis like p icture retinitis,
anem ia and o th er pathology. W ith the advent o f chem otherapy for C M V 35, the precise identification o f th e v im s is o f crucial im portance, m ainly in a com prom ised host o r after tran splantation. F o r a review o f conventional diagnostic m ethods for C M V infection, see C H O U (1990) 14. T hrough m olecular biology techniques, C M V d etection can be attained by IS H in histological sections24 o r in cell sm ears30, by d ot b lo t o f u rin e 15 and by PC R from blood, urin e o r fresh o r fixed tissues58. H ow ever, a positiv e C M V P C R resu lt should b e cautiously interpreted, since C M V infections are usually o f latent form and the m ajority o f adults in the norm al p opulation are infected.
R etrovirus
R etroviruses are single-stranded R N A viruses. F ollow ing cell entry and by th e action o f a reverse transcriptase, the R N A genom e is converted to double-stranded D N A (p ro v iru s), w hich integrates into the host cellular genom e and rem ains there for the life o f the cell.
T he hum an retro v iru s so far characterized are HIV -1 and H IV -2, causative agents o f A cquired Im m unodeficiency S yndrom e (A ID S ), and H T L V -1 and H T L V -II, associated w ith T -cell leukem ias. R etrovirus infections are usually diagnosed by serologic tests, b u t som e factors lim it th e efficiency o f these m ethods3655: 1. the tran scrip tio n dorm ancy o f the proviral genom e; 2. the sm all n u m b er o f infected cells in peripheral blood; 3. the sm all num ber o f proviral copies p er infected cell; 4. in n ew borns, th e presence o f p assiv ely acq u ired m aternal antibody and the lack o f reliab le tests for H IV -specific IgM . Because o f this an d du e to its great sensi tivity, P C R has becom e the best procedure for detection o f these v iru ses36. B ecause o f the sm all num ber o f infected circu latin g cells, oth er hybridization procedures such as ISH an d S outhern blo t can give false negative results.
P C R has p ro v en to b e v ery useful fo r H IV id en tificatio n in th e fo llo w in g co n d itio n s: 1. confirm ing infection in seropositive individuals negative by v iru s culture48; 2 . infected individuals p r i o r to s e r o c o n v e r s i o n 33. C l i n i c a l a n d epidem iological evidence show s that H IV can establish a latent infection w ith low viral expression and absence o f specific antibody response. In this situation, the infected p erso n is a potential source o f
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n i q u e s f o r th e d i a g n o s i s o f i n f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
spread o f the v irus; 3. resolving the infection status w h e n r e s u lts o f th e W e s te rn b lo t assay are a m b ig u o u s ^ ; 4. new born o r seronegative children b o m to sero p o sitiv e m others39 55; 5. seronegative in dividual at h ig h -risk o f infection33 50. P C R is also effective in d eterm ining th e type o f the virus51.
O ther v iru ses
In a lesser frequency, m olecular hybridization techniques are also being applied to detection o f adenoviruses, enteroviruses, H e rp e s s im p le x v im s72, E pstein B arr v iru s63 and ru bella v iru s32.
B A C T E R IA L D ISEA SES
M o lecu lar hybridization technology for the diagnosis o f b acterial disease is still less than for v iral infection. H ow ever, th e potential usefulness o f this m ethodelogy is clearly dem onstrated in certain conditions. T h e bacteria m ost studied w ith this approach are listed below .
H e l i c o b a c t e r
M an y su c c essfu l m o le c u la r h y b rid iz a tio n p r o c e d u r e s a re n o w a v a ila b le fo r d e te c tin g H e l i c o b a c t e r p y l o r i . T he m ost used are IS H o r dot b lo t in g astric biopsies66. P C R perform ed in fresh tissues o r in gastric aspirate specim ens can detect very low num bers o f bacteria (1 00 o r m ore per sam ple)65. W ith such sensitivity, P C R is also w ell- suited fo r v erify in g eradication fo llo w in f therapy. T h e nex t step seem s to the use o f P C R in m ore accessible sam ples, such as saliva, bool or feces.
C a m p y l o b a c t e r
M any C a m p y l o b a c t e r species have em erged as im p o rtan t causes o f gastrointestinal disease their clinical picture being indistinguishable from illnesses associated oth er enteropathogens. Conventional identification o f distin ct species o f these bacteria is troublesom e and m ore practical and accurate methods are h ig h ly desired. H y b ridization techniques w ith D N A p ro b es, including non-radioactive o n es12, can p ro v id e results com parable to culture and r e p r e s e n t a v i a b l e a l t e r n a t iv e f o r r o u tin e ap plication61.
E s c h e r i c h i a c o l i
F o r each E . c o l i strain o f m edical interest there
are specific D N A probes25 44 46, w h ich belong to two categories: cloned genes linked to one phenotype (e.g . o n e toxin) o r syn th etic o lig o n u cleo tid es hom ologous to specific sequences.
T he m ost com m om use o f D N A probes fo r the diagnosis o f these b acteria is colony h ybridization, in w hich bacterial colonies are directly transferred to filte rs an d s u b s e q u e n tly h y b r id is e d . T h e specificity and sensitivity o f th e m ethod are great and its perform ance is sim pler than conventional procedures, which, as a rule, are expensive, difficult do p erfo rm an d u su ally lim ited to re fere n ce laboratories. F o r all th ese reaso n s, m o lecu lar hybridization for detecting differen t strains o f E . c o li has represented a real advance in clinical diagnosis and for epidem iological studies.
E . c o li detection can also b e m ade by P C R , w hich recognises one single bacterium p er reaction47. Besides their use in clinical sam ples, th ere is also great interest in the ap plication o f all these tests in detection o f such bacteria in w ater5 o r p o tentially contam inated food18.
S h i g e l l a
E n te ro c y te in v a s io n b y S h i g e l l a a n d by enteroinvasive E . c o l i (E IE C ) is essential for the appearence o f clinical sym ptom s o f the infection. D N A probes correspondent to on e segm ent o f the p la s m id r e s p o n s ib le f o r in v a s io n r e c o g n is e efficiently v irulent S h i g e l l a and E IE C strains25 67. O n th e other hand, the identification o f a particu lar sequence w ithin this plasm id m ade p o ssib le the d ev e lo p m en t o f a P C R a m p lific a tio n sy stem substantially m ore sensitive than o th er tests22. Its m ajor disadvantage is that it is u n ab le to identify precisely the bacterial type, since it recognises indistinctively E IE C and various S h i g e l l a species.
M ycobacteria
D ifferen t types o f m ycobacteria are im port c a u s e o f b o th m o r b i d i ty a n d m o r t a l i t y . M y c o b a c te r iu m t u b e r c u lo s is and other m ycobacteria species cause pulm onary diseases w hich are clinically very sim ilar. T herapeutic agents h av e to b e d istinct for each type o f bacterium , so the kno w led g e o f the etiologic agent is crucial fo r treatm ent. A ccurate identification o f the m any types o f m ycobacteria by co n v en tio n a l m eth o d s is tim e -c o n su m in g a n d difficult.
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l t e c h i n i q u e s f o r t h e d i a g n o s i s o f in f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
D N A probes fo r detecting m ycobacteria o f m edical im p o rtan ce by dot b lotting are already available and w o rk w ell49 54. A m ore prom issing strategy is P C R am plification o f sequences conserved in all m ucobacteria and hybridization o f the amplified p ro d u ct w ith specific pro b es for M . t u b e r c u l o s i s , M . a v i u m - i n t r a c e l l u l a r e and M . f o r t u i t u m 1. W ith this technique ten o r m ore bacilli p er sam ple can be detected.
A system fo r detecting M . le p r a e by P C R has been d ev ised , by w h ich few bacteria can b e recognized27. T his seems to b e very im portant not o nly fo r id entifying M . l e p r a e in paucibacillary leprosy b u t also fo r distinguishing this agent from o th er m ycobacteria in skin.
P A R A SIT IC D ISEA SES
T he use o f techniques o f m olecular biology for diagnosing parasitic diseases is still in its initial phase b u t fo r som e parasites significant progress has already been achieved.
E n t a m o e b a h i s t o l y t i c a
O f the 500 m illion people infected by E ,
h i s t o l y t i c a w o rld w id e, only about 10% develop clinically o v ert disease. D ifferences in virulence am ong E . h i s t o l y t i c a isolates seem to be im portant for indu cin g lesions and sym ptom s, since there are p athogenic and non-pathogenic strains.
T he diagnosis o f E . h is t o l y t i c a in clinical samples can de d one fast and accurately by dot blot56. R ecen tly , D N A specific probes for pathogenic and non-pathogenic strains w ere obtained from tandem ly repeated sequences present in extrachrom osom al D N A , w hich can detect the parasite w ith good
sensitivity7 10.
By virtu e o f differences in restricito n pattern o f certain genes in pathogenic and non-pathogenic strains, the am plification o f these sequences by P C R and the subsequent electrophoresis analysis o f the fragm ents resulted from d ig estio n by restriction endonucleases allow the d istinction o f the strains w ith even greater efficiency60. By this m ethod, 10 o r m ore parasites can be recognized in a sam ple.
O ther parasites
T hrough the use o f total genom ic D N A as probe, som e authors have used d o t blo ttin g for d etectin g T r y p a n o s o m a c r u z i in b lo o d 3. T h e am plification o f a T. c r u z i highly repetitive D N A sequence by P C R perm its the identification o f a single parasite in a sam ple45. W ith D N A probes d eriv ed fro m m ito c h o n d ria l k in e to p la s t D N A (kD N A ), species and subespecies o f L e i s h a m a n i a can be identifyed by blots o r by IS H 4. A m plification o f kD N A sequences by P C R im proves considerably the detection o fL e is h m a n ia. M any detection system s o f P l a s m o d i u m, especially P . f a l c i p a r u m , can be perform ed for the diagnostic o f m alaria 4, P , f a l c i p a r u m can still be identifyed by P C R 34. T he
use o f P C R for the diagnosis o f giard iasis seem s to be p rom ising, since sm all num bers o f th e parasite (around 103 G i a r d i a l a m b l i a p er sam ple) can be recognized11. So, com pared to o th er diagnostic m ethods, P C R represents a real advance in the d ia g n o s tic o f g ia r d ia s is , T h e in f e c tio n by T o x o p la s m a g o n d i i can b e accurately diagnosed by P C R 27, c o n s titu tin g a p r o g r e s s o v e r o th e r identification tests fo r this parasite. F o r m etazoa, specific probes are available fo r T a e n ia s o l i u m53 and T a e n ia s a g i n a t a 21 and are useful fo r diagnosing these parasites through S outhern o r d o t b lo tting.
R EFE R E N C ES
1. A lte r H J, P u r c e ll R H , S h ih J W , M e lp o ld e r JC ,
H o u g h to n M , C h o o Q -L , K u o G . D e te c tio n o f
an tib od y to hepatitis C viru s in p r o sp ectiv ely fo llo w ed
tr a n sfu sio n r ecip ien ts w ith a c u te and c h r o n ic n o n -A ,
n o n -B h e p a titis . N e w E n g la n d Jou rn al o f M e d ic in e
3 2 1 : 1 4 9 4 - 1 5 0 0 , 1 9 8 9 .
2 . A r e n s M , S w ie r k o s z E M . D e te c tio n o f rotavirus b y
h y b r id iz a tio n w ith a n o n r a d io a c tiv e sy sn th e tic D N A
p r o b e an d c o m p a r iso n w ith c o m m e r c ia l e n s y m e
im m u n o a ssa y s an d silv e r -s ta in e d p o ly a c r y la m id e
g e ls . Jou rn al o f C lin ic a l M ic r o b io lo g y 2 7 :1 2 7 7
-1 2 7 9 , -1 9 8 9 .
3 . A sh a ll F , Y iu p -C h u c k D A M , L u q u etti A A , M ile s
A M . R a d io la b e le d to ta l p a r a s ite D N A p r o b e
sp ecifica lly d etec ts T r y p a n o s o m a c r u z i in m a m m alian b lo o d . J ou rn al o f C lin ic a l M ic r o b io lo g y 2 6 : 5 7 6
-5 7 8 , 1 9 8 8 .
4 . B arle r Jr. R H . D N A p r o b e d ia g n o s is o f p a ra sitic
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n i q u e s f o r th e d i a g n o s i s o f in f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
in fe c tio n s . E x p e r im e n ta l P a r a sito lo g y 7 0 :4 9 4 - 4 9 9 ,
1 9 9 0 .
5 . B ej A K , D iC e s a r e J L , H a f f L , A tla s R M . D ete c tio n
o f E s c h e r i c h i a c o l i and S h i g e l l a s s p . in w a te r y u sin g th e p o ly m e r a se c h a in rea ctio n and g e n e p r o b e s for
u i d . A p p lie d a n d E n v ir o n m e n ta l M ic r o b io lo g y 5 7 : 1 0 1 3 - 1 0 1 7 , 1 9 9 1 .
6 . B o p p C A , T h re att V L , M o s e le y S L , W e lls JG ,
W a c h s m u t h I K . A c o m p a r i s o n o f a lk a l in e
p h o sp h a ta s e an d r a d io la b e lle d g e n e p r o b e s w ith
b io a s s a y s fo r e n te r o to x ig e n ic E s c h e r i c h i a c o l i . M o le c u la r and C ellu la r P ro b es 4 :1 9 3 - 2 0 3 , 1 9 9 0 .
7 . B ra ch a R , D ia m o n d L S , A c k e r s JP, B urchard G D ,
M ir e lm a n D . D iffe r e n tia tio n o f c lin ic a l is o la te s o f
E n t a m o e b a h is t o l y t i c a b y u sin g s p e c ific D N A p r o b e s . Jo u rn a l o f C lin ica l M ic r o b io lo g y 2 8 : 6 8 0 - 6 8 4 ,1 9 9 0 .
8 . B r e c h o t C , D e g o s F , L u g a ss y C , T h ie r s V , Z afra ni
S , F ra n co D , B ism u ty H , T rep o C , B en h a m o u J-P , W a n d s J, Is se lb a c h e r K , T io llä is P , B erth elo t P.
H ep a titis B v ir u s in p atien ts w ith c h r o n ic liv e r
d is e a s e and n e g a tiv e te sts fo r hep atitis B su rfa ce
a n tig e n . N e w E n g la n d Journal o f M e d ic in e 3 1 2 :2 7 0 -2 7 6 , 1 9 8 5 .
9 . B r is s o n N o e l A , G ic q u e l B , L e c o s s ie r D , L e v y
-F reb a u lt V , N a s s i f X , H a n c e A j. R a pid d ia g n o s is o f
tu b e r c u lo sis b y a m p lifica tio n o f m y co b a c te r ia l D N A
in c lin ic a l s a m p le s. L a n c e t 1 1 :1 0 6 9 -1 0 7 1 , 1 9 8 9 .
1 0 . B u rch D J , L i E , R e e d S , J a c k so n T F H G , Sta n ley Jr.
S L . Isolation o f a strain-specific E n ta m o e b a h is t o ly t ic a c D N A c lo n e . Jo u rn a l o f C lin ic a l M ic r o b io lo g y
2 9 : 6 9 6 - 7 0 1 , 1 9 9 1 .
1 1 . C ar n a b y S , M c H u g h T D , H a ll S , B u tc h er P D ,
F a rth in g M JG . P o ly m e r a se ch a in rea ctio n d ia g n o s is
o f G i a r d i a l a m h l i a in m a n . G a s tr o e n te r o lo g y 1 0 0 :A 5 6 5 , 1 9 9 1 .
1 2 . C h e v r ie r D , L a rzu l D , M eg ra u d F , G u esd o n J-L .
Id en tifica tio n an d c la s sific a tio n o f C a m p y l o b a c t e r stra in s b y u s in g n o n r a d ia c tiv e D N A p r o b e s. Jou rna l
o f C lin ic a l M ic r o b io lo g y 2 7 .3 2 1 - 3 2 6 , 1 9 8 9 .
1 3 . C h o o Q -L , K u o G , W e in e r A J , O v e r b y L R , B r a d le y D W , H o u g h to n M . Iso la tio n o f a c D N A c lo n e
d e r iv e d fr o m a b lo o d -b o r n e n o n -A , n o n -B vira l
h ep a titis g e n o m e . S c ie n c e 2 4 4 : 3 5 9 - 3 6 1 , 1 9 8 9 .
1 4 . C h o u S . N e w e r m e t h o d s f o r d i a g n o s i s o f
c y to m e g a lo v ir u s in fe c tio n . R e v ie w s o f In fectio u s
D is e a s e s 1 2 (s u p p l 7 ) : S 7 2 7 - 7 3 6 , 1 9 9 0 .
1 5 . C h o u S , M e r ig a n T C . R a p id d e t e c t io n a n d
qu an tita tion o f h u m a n c y to m e g a lo v ir u s in urine
th ro u g h t D N A h y b r id iz a tio n . N e w E n g la n d Journal
o f M e d ic in e 3 0 8 : 9 2 1 - 9 2 5 , 1 9 8 3 .
16. D e n n isto n K J, H o y e r B H , S m e d ile A , W e lls F V ,
N e ls o n J , G erin JL . C lo n ed fr a g m en t o f th e h epa titis
d elta viru s R N A g e n o m e : S e q u e n c e and d ia g n o s is
ap p lica tio n . S c ie n c e 2 3 2 .8 7 3 - 8 7 5 , 1 9 8 6 .
1 7. D im itr o v D H , G rah am D Y , E s te s M K . D e te c tio n o f
ro ta v iru se s b y n u c le ic a cid h y b rid isa tio n w ith c lo n e d
D N A o f sim ia n rota viru s S A 1 1 g e n e s . Journ al o f
In fectio u s D is e a s e s 1 5 2 : 2 9 3 - 3 0 0 , 1 9 8 5 .
1 8. D o v e y S , T o w n e r KJ. A b io tin y la te d D N A p r o b e to
d e te ct b a cte ria l c e lls in a r tific ia lly co n ta m in a te d
fo o d s tu ffs . J ou rn al o f A p p lie d B a c te r io lo g y 6 6 :4 3 -4 7 , 1 9 8 9 .
1 9 . E id e n JJ, F ir o o z m a n d F , S a to S , V o n d e r fe c h t S L ,
Y in F Z , Y o lk e n R H . D e te c tio n o f g r o u p B rotav irus
in fe c a l sp e c im e n s b y d o t h y b rid iza tio n w ith a
c l o n e d c D N A p r o b e . J o u r n a l o f C l i n i c a l M ic r o b io lo g y 2 7 - 4 2 2 - 4 2 6 , 1 9 8 9 .
2 0 . F a rci A , W o n g D , A lte r H J, M ille r R H , S h ih JW ,
P u r c e ll R H . D e te c tio n o f H C V s e q u e n c e s b y P C R
in hepatitis C viru s in fec tion : rela tio n sh ip to an tib o d y
r e s p o n s e and c lin ic a l o u tc o m e . H e p a to lo g y 1 2 :9 0 4 , 1 9 9 0 .
2 1 . F lis s e r A , R eid A , G r a c ia -Z e p e d a E , M c m a n u s D P .
S p e c ific d e te c tio n o f T a e n ia s a g i n a t a e g g s b y D N A h y b rid iza tio n . L a n c e t 1 1 :1 4 2 9 -1 4 3 0 , 1 9 8 8 .
2 2 . F ra n k el G , R ile y L , G iro n J A , V a lm a s s o i J,
F ried m an n A , S tr o c k b in e N , F a lk o w S , S c h o o ln ik
G K . D e te c tio n o f S h i g e l l a in fe c e s u sin g D N A a m p lific a tio n . J o u r n a l o f I n f e c t io u s D i s e a s e s
1 6 1 :1 2 5 2 - 1 2 5 6 , 1 9 9 0 .
2 3 . G a r so n J A , T e d d e r R S , B r ig g s M , T u k e P ,
G la z e b r o o k J A , T ru te A , P ark er D , B arbara JA J,
C on trera s M , A lo y s iu s S . D e te c tio n o f h ep atitis C
v i r a l s e q u e n c e s in b l o o d d o n a t i o n s b y
“n e s te d ’’p o ly m e r a s e ch ain re a ctio n and p red ic tio n
o f in fe c tiv ity . L a n cet 3 3 5 : 1 4 1 9 - 1 4 2 2 , 1 9 9 0 .
2 4 . G nann Jr JW , A h lm e n J, S v a la n d e r C , O ld in g L ,
O ld sto n e M B A , N e ls o n J A . In fla m m a to ry c e lls in
tr a n s p la n te d k id n e y s a r e in f e c t e d b y h u m a n
c y to m e g a lo v ir u s . A m e r ic a n Jo u rn a l o f P a th o lo g y 1 3 2 :2 3 9 - 2 4 8 , 1 9 8 8 .
2 5 . G o m e s T A T , T o le d o M R F ,T r a b u ls iL R ,W o o d P K ,
M o rris Jr JG . D N A p r o b e s fo r id e n tific a tio n o f
e n te r o in v a s iv e E s c h e r i c h i a c o l i . J ou rn al o f C lin ic a l M ic r o b io lo g y 2 5 : 2 0 2 5 - 2 0 2 7 , 1 9 8 7 .
2 6 . G riffiths P D , G ru n d y J E . M o le c u la r b io lo g y and
im m u n o lo g y o f c y t o m e g a lo v ir u s . B io c h e m ic a l Jou rn al 2 4 1 : 3 1 3 - 3 2 4 , 1 9 8 7 .
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l t e c h i n i q u e s f o r th e d i a g n o s i s o f in f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
2 7 . G r o v e r C M , T h u llie z P , R em in g to n J S , B oo th ro y d
J C . R a p id p r e n a t a l d ia g n o s i s o f c o n g e n it a l
T o x o p l a s m a in fe c tio n b y u sin g p o ly m e r a s e chain r ea ctio n and a m n io tic flu id . Journ al o f C lin ica l
M ic r o b io lo g y 2 8 : 2 2 9 7 - 2 3 0 1 , 1 9 9 0 .
2 8 . G u im a rã es E M , B ra sileir o F ilh o G , P ena S D J . H u m a n p a p illo m a v ir u s d e t e c t io n in c e r v ic a l
d y s p la s ia s o r n e o p la s ia s an d in c o n d y lo m a ta
acu m in a ta b y in s i t u h y b rid iza tio n w ith b io tiny la ted D N A p r o b e s. R e v ista do Instituto d e M ed ic in a
T r o p ic a l (in p r e s s ).
2 9 . H a c k e l C , H o u a rd S , P o rta els F , V a n E lse n A ,
H e r z o g A , B o lle n A . S p e c ific id en tifica tio n o f
M y c o b a c t e r i u m l e p r a e b y th e p o ly m e r a s e chain r e a c tio n . M o le c u la r and C ellu la r P ro b es 4 :2 0 5 - 2 1 0 ,
1 9 9 0 .
3 0 . H ilb o m e L H , N ie b e r g R K , C h e n g L , L e w in KJ.
D ir e c t in s i tu h y b rid iza tio n fo r rapid d ete ctio n o f c y t o m e g a l o v i r u s in b r o n c h o a lv e o l a r la v a g e .
A m e r ic a n J our na l o f C lin ic a l P a th o lo g y 8 7 :7 6 6
-7 6 9 , 1 9 8 -7 .
3 1 . H o p k in J M , W a k e fie ld A E . D N A h y b rid iza tio n for
th e d ia g n o s is o f m ic r o b ia l d is e a s e . Q u artely Journal
o f M e d ic in e 2 7 7 : 4 1 5 - 4 2 1 , 1 9 9 0 .
3 2 . H o - T e r r y L , T e r r y G M , L o n d e s b o r o u g h P.
D ia g n o s is o f fo e ta l r u b e lla v ir u s in fe c tio n b y
p o ly m e r a s e c h a in re a c tio n . Jou rn al o f G en er al
V ir o lo g y 7 1 : 1 6 0 7 - 1 6 1 1 , 1 9 9 0 .
3 3 . Im a g a w a D T , L e e M H , W o lin sk y S M , S a n o K ,
M o r a le s F , K w o k S , S n in s k y JJ, N ish a n ia n PG , G io r g i J , F a h e y J L , D u d le y J , V is s c h e r B R , D e te ls
R . H u m a n im m u n o d e fic ie n c y v ir u s ty p e 1 in fectio n
in h o m o s e x u a l m en w h o rem ain s e r o n e g a tiv e for
p ro lo n g ed p erio d s. N e w E nglan d Journal o f M ed icin e
3 2 0 : 1 4 5 8 - 1 4 6 2 , 1 9 8 9 .
3 4 . J a u reg u ib erry G , H a tin I, d ’A u r io l L , G a lib er t G.
P C R d e te c t io n o f P l a s m o d i u m f a l c i p a r u m b y o lig o n u c le o t id e p r o b e s. M o le c u la r and C ellu la r
P r o b e s 4 : 4 0 9 - 4 1 4 , 1 9 9 0 .
3 5 . J ia n g X , E ste s M K , M e tc a lfT G . In s i tu hybridizatio n fo r q u a n tita tiv e a s s a y o f in fe c tio u s hep atitis A v ir u s .
Jou rn al o f C lin ic a l M ic r o b io lo g y 2 7 : 8 7 4 - 8 7 9 ,1 9 8 9 . 3 6 . K w o k S , S n in s k y JJ. A p p lic a tio n o f P C R to th e
d e te c tio n o f h u m a n in fe c tio u s d is e a s e s . In : E rlich H A ( e d ) . P C R T e c h n o l o g y . P r in c i p le s a n d
A p llic a tio n s fo r D N A A m p lifica tio n . Sto ck to n P ress, N e w Y o r k p .2 3 5 - 2 4 4 , 1 9 8 9 .
3 7 . L a r z u l D , G u ig u e F , S n in s k y JJ, M a c k D H , B réch o t
C , G u e s d o n J -L . D e te c tio n o f hep atitis B viru s
se q u e n c e s in se ru m b y u sin g in v i t r o e n z y m a tic a m p lific a tio n . J o u r n a l o f V ir o lo g ic a l M e th o d s
2 0 :2 2 7 - 2 3 7 , 1 9 8 8 .
3 8 . L a sk in O L , S ta h l-B a y liss C M , K alm an C M , R o se c a n
L R . U s e o f g a n c i c l o v i r to t r e a t s e r i o u s
c y to m e g a lo v ir u s in fe c tio n s in p a tien ts w ith A ID S .
Jou rn al o f In fectio u s D is e a s e s 1 5 5 : 3 2 3 - 3 2 6 , 1 9 8 7 . 3 9 . L a u re F , C o u rg n a u d V , R o u z io u x C , B la n c h e s S ,
V e b e r F , B urgard M , J a c o m e t C , G r isc e lli C ,
B rech o t C . D e te c tio n o f H IV 1 D N A in infan ts and
ch ild ren b y m ea n s o f t h e p o ly m e r a s e ch a in rea ctio n .
L a n c e t 1 1 :5 3 8 -5 4 1 , 1 9 8 8 .
4 0 . L e h n H , V illa L L , M a r z io n a F , H ilg a rth M , H illem an s H -G , S a u er G . P h y sica l state and b io lo g ic a l
a c tiv ity o f h u m a n p a p illo m a v ir u s g e n o m e s in
p r e c a n c e r o u s le s io n s o f th e fe m a le g e n ita l tract.
Journ al o f G en era l V ir o lo g y 6 9 : 1 8 7 - 1 9 6 , 1 9 8 8 .
4 1 . L o r in c z A T , T e m p le G F , K urm an R J, J e n so n A B ,
L a n ca ste r W D . O n c o g e n ic a s s o c ia tio n o f s p e c ific
hu m an p a p illo m a v ir u s ty p e s w ith c e r v ic a l n e o p la s ia .
J o u r a n lo f th e N a tio n a lC a n c e r In stitu te 7 9 : 6 7 1 - 6 7 7 ,
1 9 8 7 .
4 2 . M e lc h e r s W , B ru le A , W a lb o o m e r s J , B ru in M ,
B u r g e r M , H e r b r in k P , M e ij e r C , L in d em a n J, Q uint W . In crea sed d e te c t io n r a te o f hu m a n p a p illo m a v ir u s
in c e r v ic a l sc r a p e s b y th e p o ly m e r a s e c h a in rea ctio n
a s co m p a red to m o d ifie d F IS H an d S o u th ern -b lo t
a n a ly s is. Jou rn al o f M e d ic a l V ir o lo g y 2 7 : 3 2 9 - 3 3 5 ,
1 9 8 9 .
4 3 . M iy a m u ra T , S aito I, K ata ya m a T , K ik u ch i S , T ateda A , H o u g h to n M , C h o o Q -L , K uo G . D ete c tio n
o f an tib od y a g a in st antigen e x p r e s s e d b y m o le c u la r ly
c lo n e d h ep atitis C v ir u s c D N A : a p p lic a tio n to
d ia g n o s is and b o o l s c r e e n in g fo r p o sttra n sfu sio n
h ep a titis. P r o c e e d in g s o f th e N a tio n a l a c a d e m y o f
S c ie n c e s o f th e U n ite d S ta tes o f A m e r ic a 8 7 : 9 8 3
-9 8 7 , 1 -9 -9 0 .
4 4 . M o s e le y S L ,E c h e v e r r ia P ,S e r iw a t a n a J ,T ir a p a t C ,
C h a ic u m p a W , S a k u ld a ip e a r a T , F a lk o w S .
Id en tifica tio n o f E n te r o to x ig e n ic E s c h e r i c h i a c o l i b y c o lo n y h y b rid iza tio n u sin g th r e e e n te r o to x in g e n e p r o b e s . Jou rn al o f In fectio u s D is e a s e s 1 4 5 :8 6 3
-8 6 9 , 1 9 -8 2 .
4 5 . M o s e r D R , K ir c h h o ffL V , D o n e ls o n J E . D e te c tio n
o f T r y p a n o s o m a c r u z i b y D N A a m p lific a tio n u sin g th e p o ly m e r a s e c h in r ea ctio n . Jo u rn a l o f C lin ic a l
M ic r o b io lo g y 2 7 : 1 4 7 7 - 1 4 8 2 , 1 9 8 9 .
4 6 . N ataro J P , B a ld in i M M , K ape r J B , B la c k R E , B ra v o
N , L e v in e M M . D e te c tio n o f a n a d h e r e n c e fa cto r o f
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n i q u e s f o r t h e d i a g n o s i s o f in f e c ti o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
e n te r o p a th o g e n ic E s c h e r i c h i a c o l i w ith a D N A p r o b e . Jo u rn a l o f In fe c tio u s D is e a s e s 1 5 2 :5 6 0 - 5 6 5 ,
1 9 8 5 .
4 7 . O liv e D M . D e te c tio n o f e n te r o to x ig e n ic E s c h e r i c h ia c o l i a fter p o ly m e r a s e c h a in re a ctio n a m p lifica tio n w ith a th e r m o sta b le D N A p o ly m e r a se . Journ al o f
C lin ic a l M ic r o b io lo g y 2 7 : 2 6 1 - 2 6 5 , 1 9 8 9 .
4 8 . O u C -Y , K w o k S , M it c h e ll S W , M a c k D H , S n in sk y
JJ, K re b s J W , F e o r in o P , W a r fie l D , S ch o ch etm a n
G . D N A a m p lific a tio n fo r d ir ect d ete ctio n o f H IV
-1 in D N A o f p e r ip h era l b lo o d m o n o n u c le a r c e lls .
S c ie n c e 2 3 9 : 2 9 5 - 2 9 7 , 1 9 8 8 .
4 9 . P ao C C , L in S -S , W u S -Y , Ju an g W -M . T h e
d e te c tio n o f m y c o b a c te r ia l D N A s e q u e n c e s in
u n c u lt u r e d c l i n i c a l s p e c i m e n s w it h c lo n e d
M y c o b a c t e r i u m t u b e r c u l o s i s D N A a s p r o b e s . T u b e r c le 6 9 : 2 7 - 3 6 , 1 9 8 8 .
5 0 . P e z e lla M , R o s s i P , L o m b a rd i V , G e m e lli V , M a ria n i C o sta n tin i R , M ir o lo M , F u nd aro C ,
M o s c h e s e V , W ig z e ll H . H IV v ir a l se q u e n c e s in
se r o n e g a tiv e p e o p le at risk d etected b y in s i tu h y b rid iza tio n and p o ly m e r a s e ch ain re a ctio n . B ritish M e d ic a l J ou rn al 2 9 8 : 7 1 3 - 7 1 6 , 1 9 8 9 .
5 1 . R a y fie ld M , D e C o c k K , H e y w a r d W , G o ld ste in L ,
K reb s J , K w o k S , L e e S , M c c o r m ic k J , M o rea u J M ,
O d e h o u r i K , S c h o c h e tm a n G , S n in sk y J, O u C -Y .
M ix e d h u m a n im m u n o d e f ic ie n c y v ir u s (H IV )
in fe c tio n in an in d iv id u a l: d em o n str a tio n o f both
H IV ty p e 1 and ty p e 2 p ro v ir a l se q u e n c e s b y u sin g
p o ly m e r a s e c h a in r ea ctio n . J ou rn al o f In fectio u s
D is e a s e s 1 5 8 :1 1 7 0 - 1 1 7 6 , 1 9 8 8 .
5 2 . R e y e s G R , P u rd y M A , K im JP , L u k K -C , Y o u n g
L M , F ry K E , B r a d le y D W . Iso la tio n o f a c D N A
from th e v ir u s r e s p o n s ib le fo r e n te r ic a lly transm itted
n o n -A , n o n -B h ep a titis. S c ie n c e 2 4 7 : 1 3 3 5 - 1 3 3 9 ,
1 9 9 0 .
5 3 . R ish i A K , M c M a n u s D P . D N A p r o b e s w h ic h
u n a m b ig o u sly d is tin g u ish T a e n ia s o l i u m from T. s a g i n a t a . L a n c e t 1 1 :1 2 7 5 -1 2 7 6 , 1 9 8 7 .
5 4 . R o b e r ts M C , M c m illa n C , C o y le M B . W h o le
c h r o m o s o m a l D N A p r o b e s fo r rapid id en tifica tio n
o f M y c o b a c t e r i u m t u b e r c u l o s i s and M y c o b a c t e r i u m a v i u m c o m p le x . J ou rn al o f C lin ic a l M ic r o b io lo g y 2 5 : 1 2 3 9 - 1 2 4 3 , 1 9 8 7 .
5 5 . R o g e r s M F , O u C -Y , R a y fie l M , T h o m a s P A ,
S c h o e n b a u m E E , A b ra m s E , K ra sin ki K , S e lw y n
P A , M o o r e J , K au l A , G rim m K T , B am ji M ,
S c h o c h e t m a n G a n d T h e N e w Y o r k C it y
C o lla b o r a tiv e S tu d y o f M a te rn a l H IV T ra n sm issio n
and M o n te fio r e M e d ic a l C e n te r H I V P er in ata l T r a n sm issio n S tu d y G ro u p . U s e o f th e p o ly m e r a se
c h a in re a ctio n fo r e a r ly d e te c tio n o f th e p ro v ir a l
s e q u e n c e s o f hu m a n im m u d e fic ie n c y vir u s in infants
b o m to s e r o p o s itiv e m o th e r s. N e w E n g la n d Journa l o f M e d ic in e 3 2 0 : 1 6 4 9 - 1 6 5 4 , 1 9 8 9 .
5 6 . S a m u e lso n J , A cu n a -S o to R , R e e d S , B ia g i F , W irth
D . D N A h y b rid iza tio n p r o b e fo r c lin ic a l d ia g n o s is
o f E n t a m o e b a h i s t o l y t i c a . J o u r n a l o f C lin ic a l M ic r o b io lo g y 2 7 : 6 7 1 - 6 7 6 , 1 9 8 9 .
5 7 . S e y d a M , S c h e e le T , N eu m a n n R , K r u e g e r G R F .
C o m p a ra tiv e e v a lu a tio n o f n o n -r a d io c tiv e in s i tu h y b rid iza tio n te c h n iq u e s fo r p a th o lo g ic d ia g n o s is o f vir a l infection. Pathology R esearch and Practice
184:18-2 6 , 19 89 .
5 8 . S h ib a ta D . D e te c tio n o f hu m an c y to m e g a lo v ir u s. In : Innis M A , G elfa n d D H , S n in s k y JJ, W h ite TJ (e d ).
P C R P r o t o c o l s : A G u i d e to M e t h o d s a n d
A p p lic a tio n s. A c a d e m ic P r e s s , N e w Y o rk p .3 6 8 -3 7 1 , 1 9 9 0 .
5 9 . Sn in jd ers PJF, V a n D e n B r u le A J C , S ch rijn em a k ers
H F J , S n o w G , M e ije r C J L M , W a lb o o m e r s J M M .
T h e u s e o f g e n e r a l pr im ers in th e p o le m e r a se ch ain
re a ctio n p er m its th e d e te c tio n o f a b ro a d sp ectru m o f hu m an p a p illo m a v ir u s g e n o ty p e s . Jo u rn a l o f
G en era l V ir o lo g y 7 1 : 1 7 3 - 1 8 1 , 1 9 9 0 .
6 0 . T ä n n ic h E , B u r c h a r d G D . D if f e r e n t ia t io n o f
p a t h o g e n i c fr o m n o n p a t h o g e n i c E n t a m o e b a h i s t o l y t i c a b y restric tio n fr a g m en t a n a ly s is o f a s in g le g e n e a m p lifie d in v i t r o . J ou rn al o f C lin ic a l M ic r o b io lo g y 2 9 : 2 5 0 - 2 5 5 , 1 9 9 1 .
6 1 . T h o r n e G M , M a c o n e A , G o l d m a n n D A .
E n z y m a tic a lly la b e lle d n u c le ic a c id (N A ) p r o b e
a ss a y s fo r d e te c tio n o f C a m p y l o b a c t e r s p in hu m an fa e c a l sp e c im e n s and in cu ltu re. M o le c u la r
and C ellu la r P ro b es 4 : 1 3 3 - 1 4 2 , 1 9 9 0 .
6 2 . T in g Y , M a n o s M M . D e te c tio n and ty p in g o f g e n ita l
hu m an p a p illo m a v ir u se s . In : Innis M A , G elfa n d D H , S n in s k y JJ, W h ite TJ (e d ). P C R P r o to c o ls: A
G u id e to M e th o d s and A p p lic a tio n s. A c a d e m ic
P r e s s , N e w Y o r k p .3 5 6 - 3 6 7 , 1 9 9 0 .
6 3 . U h a ra H , S a to Y , M u k a i K , A k a o I, M a tsu n o Y ,
Fu ru ya S , H o sh ik a w a T , S h im o sa to Y , S a id a T .
D e te c tio n o f E p ste in B a rr v ir u s D N A in R e e d
-S te m b e r g c e lls o f H o d g k in ’s d is e a s e u sin g th e
p o ly m e r a s e c h a in rea ctio n and in s i t u h y b rid iza tio n . J a p a n e se J ou rn al o f C a n c e r R e s e a r c h 8 1 : 2 7 2 - 2 7 8 , 1 9 9 0 .
6 4 . U lr ic h P P , B h a t R A , S e to B , M a c k D , S n in s k y J,
A r t i g o d e R e v i s ã o . F il h o G B , P e n a S D J . M o l e c u l a r b i o l o g i c a l te c h i n i q u e s f o r t h e d i a g n o s i s o f in f e c t i o n s d i s e a s e s . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 5 : 5 9 - 7 1 , j a n - m a r , 1 9 9 2 .
V y a s G N . E n z y m a tic a m p lifica tio n o f hepatitis B
v ir u s D N A in seru m co m p a r e d w ith in fe c tiv ity in
c h im p a n z e e s . J o u r n a lo f In fectiou s D is e a s e s 1 6 0 :3 7
-4 3 , 1 9 8 9 .
6 5 . V a le n tin e J L , A rth u r R R , M o b le y H L T , D ic k JD . D e te c tio n o f H e l i c o b a c t e r p y l o r i b y u sin g th e p o ly m e r a s e c h a in r e a c tio n . J our na l o f C lin ic a l
M ic r o b io lo g y 2 9 : 6 8 9 - 6 9 5 , 1 9 9 1 .
6 6 . V a n D e n B e r g F M , Z ijlm a n s H , L a n g e n b e r g W ,
R a u w s E , S c h ip p e r M . D e te c tio n o f C a m p y l o b a c t e r p y l o r i in sto m a c h tis s u e b y D N A in si tu hybridization. J ou rn al o f C lin ic a l P a th o lo g y 4 2 : 9 9 5 - 1 0 0 0 ,1 9 8 9 .
6 7 . V a n D e r P o e l C L , R e e sin k H W , S c h a a sb e r g W ,
L e e n tv a r r -K u y p e r s A , B ark er E , E x e l-O e h le r s PJ, L e lie P N . In fe c tiv ity o f b lo o d s e r o p o s itiv e for
h ep a titis C v ir u s a n tib o d ie s. L a n c e t 3 3 5 :5 5 8 - 5 6 0 ,
1 9 9 0 .
6 8 . W ic k e n d e n C , S te e le A , M a lc o lm A D B , C o le m a n
D V . S c r e e n in g fo r w a rt viru s in fectio n in norm al
and a b n o rm a l c e r v ic e s b y D N A h y b rid iza tio n o f
c e r v ic a l s c r a p e s . L a n c e t 1 :6 5 -6 7 , 1 9 8 5 .
6 9 . W o o d P K , M o rris Jr. J G , S m a ll P L C , S eth ab u tr O ,
T o le d o M R F , T ra b u lsi L , K ap er J B . C o m p a r is o n o f
D N A p r o b e s and th e S e r e n y te s t fo r id en tifica tio n o f
in v a s iv e S h ig e lla and E s c h e r i c h ia c o l i stra in s. Journal o f C lin ic a l M ic r o b io lo g y 2 4 : 4 9 8 - 5 0 0 , 1 9 8 6 .
7 0 . Z h e n g B J , L a m W P , Y a n Y K , L o S K F , L u n g M L ,
N g M H . D ir e c t id en tifica tio n o f s e r o ty p e s o fn a tu r a l
hu m a n rota viru s is o la te s b y h y b rid iza tio n u sin g
c D N A p ro b es d eriv ed from s e g m e n t 9 o f th e rotavirus
g e n o m e . Jou rn al o f C lin ic a l M ic r o b io lo g y 2 7 : 5 5 2
-5 -5 7 , 1 9 8 9 .
7 1 . Z ig n e g o A L , D e n y P , F e r a y C , P o n zetto A , G en tilin i
P , T io lla is P , B r e c h o t C . A m p lific a tio n o f h ep a titis d elta v ir u s R N A s e q u e n c e s b y p o ly m e r a s e ch ain
reaction : a to o l fo r v ir a l d e te c tio n an d c o in in g .
M o le c u la r C ellu la r P r o b e s 4 : 4 3 - 5 1 , 1 9 9 0 . 7 2 . Z w a d y k Jr P, C o o k s e y R C . N u c le ic a cid p r o b e s in
c lin ic a l m ic r o b io lo g y . C R C C ritica l R e v ie w s in
C lin ic a l L ab o ra to ry S c ie n c e s 2 5 : 7 1 - 1 0 3 , 1 9 8 7 .
