36
Molecular Techniques
T a b le 3 6 .3 T raditional H ybridization Techniques Used fo r H um an P apillom avirus Testing
T e c h n iq u e
S e n s itiv ity (c o p ie s /c e ll)
D N A n e e d e d
L a b e l a n d te s tin g tim e
A d v a n ta g e (s )
D is a d v a n ta g e (s )
S outhern b lo t
0.1
10 pg
• 32P: 4 -5 days
• N onradioactive: 2 days
• D etects n e w types
• In te g ra te d/ep iso m al
• H ig h /lo w stringency
• Very laborious
• N ot suitable fo r
screening
D ot b lo t
1.0
500 ng
• 32P: 4 -5 days
• N onradioactive: 2 days
• H ig h /lo w stringency
• S uitable fo r screening
• Requires ind ivid u a l
labeling reactions
• H igh strin ge n cy o n ly
Reverse b lo t
1.0
500 ng
• 32P: 4 -5 days
• N onradioactive: 2 days
• H ig h /lo w stringency
• Several typ e s can be
id e n tifie d at th e sam e
tim e
• Requires ind ivid u a l
labeling reactions
Tissue in situ
2 0 -5 0
Few cells
• 35S: 6 -8 days
• N onradioactive: 1 day
• C ellular localization
• C orrelation o f HPV typ e
w ith m o rp h o lo g y
• Use o f ro u tin e ly fixed
tissues
• Type relatedness o nly
• C ross-reactivity
Filter in situ
1 -5 x 104-5 HPV D NA
m olecules
Few cells
• 32P: 1 -2 days
• N o D NA extra ctio n
• Rapid
• B ackground p roblem s
• H igh co p y n u m b e r/
cell
S andw ich
hybrid iza tio n
1 -5 x 105 HPV D NA
m olecules
105-1 0 6 cells
• 32P: 1 day
• N onradioactive: 6 h
• Rapid
• D etectio n w ith liquid
scintillatio n
• O ne ty p e /sa m p le
analyzed
Polym erase chain
reaction
1000-2000/1 pg
n o n ta rg e t DNA
300 ng
• 5 h
• Sensitive
• S em ia u to m a te d
• C o n ta m in a tio n
• Relatedness o nly
H ot-start polym erase
chain reaction
1-10/1 pg
300 ng
• 5 h
• H ighly sensitive
• Requires n o n ta rg e t
D N A sequence
in fo rm a tio n
Polym erase chain
reaction in situ
1
Few cells
• 1 day
• H ighly sensitive
• False positives
fre q u e n t
• Several co ntro ls
needed
HPV, human papillomavirus.
(After Syrjanen and Syrjanen 2000,
10
with permission of Wiley.)
the discussion o f the use o f H C 2 and PCR-based assays in H P V
detection and research, these new m ethods are introd uced in
b rie f here.
Tyram ide-Am plified In Situ Hybridization
T he se n sitivity o f c onventional
in situ h yb rid iz a tio n falls
between 20 and 50 copies per cell (Table 3 6.3 ) , w h ic h fre q u e n tly
precludes detection o f the low -cop y n um b er H P V infections in
clinical samples. D u rin g the past 10 years, several systems have
been developed to a m p lify th e h yb rid iz a tio n signal and con-
sequently im p ro ve the se n sitivity o f in situ h yb rid iza tio n . O ne
o f the m ost successful so lu tio n s has been signal a m p lific a tio n
b y tyram id e using the catalyzed rep orter d ep ositio n technique.
A t least tw o com m ercial applications are available: (1) cata-
lyzed signal a m p lifie d c o lo rim e tric D N A in s itu h yb rid iz a tio n
(G e n P o in t system, D ako, G lostrup, D e nm a rk), and (2 ) the
TSA B io tin system (P erkin Elm er, Boston, M A ). In the form er,
h yb rid iz a tio n is fo llo w e d b y p rim a ry strep tavid in -ho rse rad ish
peroxidase (H R P ), and after washing, b io tin -ty ra m id e s o lu tio n ,
fo llo w e d b y the secondary stre p ta vid in -H R P incub ation. The
TSA B io tin system also u tilize s H R P to catalyze the d ep osition
o f th e b io tin -la b e le d tyram ide. In o u r hands, the sen sitivity
o f th is m eth od seems to be one copy o f H P V per cell, i.e. the
single-copy S iH a cells give a p ositive H P V signal.
Polymerase Chain Reaction-based Methods
As explained before, PCR-based m ethod s rely o n the use o f
eith er type-specific prim ers a m p lifyin g one specific H P V type
o r consensus, general, o r m u ltip le x prim ers a m p lifyin g a broad
spectrum o f H P V genotypes. Consensus o r general prim ers tar-
get a conserved region, in m ost cases w ith in the h ig h -risk H P V
L1 gene, o f d iffe re n t H P V genotypes. Three d iffe re n t designs o f
general PC R p rim e r have been generally used. T he first approach
includes one fo rw a rd and one reverse p rim e r aim ed at a con-
served region com p lem enting to ta lly o n ly one o r a few H P V
genotypes. A n exam ple o f th is is the G P 5-p ositive/6-p ositive
PCR system .63 T he second approach uses fo rw a rd and reverse
prim ers th a t contain one o r m ore degeneracies to com pensate
fo r the intra typ ic sequence va ria tio n at the p rim in g sites. A n
exam ple o f th is is th e M Y09/11 PCR system. The th ird o p tio n is
to com b ine a n um b er o f d iffe re n t fo rw a rd and reverse prim ers.
A n exam ple o f such m u ltip le p rim e r sets are the PG M Y09/11
1083
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