An imp- K Rm ethod for detection of W-1 pmvhl DNA of a wide range 
of subtypes and recornbinant fonms amdating globally 
WeSdnera*, Uwe  asse ens b, Wolfgang G3hdec,w aiter sihwskid,[ ;eorgina Odaibo t, 
David Olaleyet, Doris Reicheftf, Burkhard GreveS 
A B S T R A C T  
rttepesemomrsionwindowpetlod(Lbomoya&~.(3) 
the reatgd%w if the W-t RplA lev& fall Mow h e d etectable 
h E 4 o o  with HRr-1 is  c h x k r k d  by the d a m of  HIY- m d w h g H A m { ~ e r l L , a O W ; p l p & & a # r z ) . A d d i -  
1 plasma RPlh w h i i  in addition tn the memk of-+ T& tioMUy, U x  W o a of pmvints DNA muld be a rsetul m a r k  
g e r w g k ~ ~ p r i m d p a l m o n i ~ ~ f w a a e s s i n g d ifsw -a  amhkg  viral and m a s s s  --term impaa 
ease ~pmgrxsahn d reepoase to anti-rmwiml therapy (m d ~ ( a m c t d . , l ~ ~ e t & Z # # ; ~ u &  
& & a o w ; c r m U 4 X m , # r a e : ~ a d ~ 2 0 0 8 :* ssrZ 
~ e t a L well~ d&gnMdPCR -bedt~sgaregble Amajorproblaninthtampli~narsdd ePectionofHlVpmvi- 
m detect and quwtify HN-1 RNA in plasma samples d M rat DNA is &e premm? d high amount of human DNA in the 
i o d i d u a I s ( ~ d a L # # 5 m. ~  a&2Mn..m, sample which ia[tuen0es tbe P[St f b c t k r m ~trh~e  mall sample 
~~IncoatrassHIV-~1D N A i s i m m o b i l I z e d i n ~ b -v ~ a f ~ ~ ~ M D C J b b d d ~ t h e ~ ~  
c r a l ~ ~ u d e a r c e l l s ( r n M t s ] a a d l y m p h o i d ~ ~cdnl~ d u r i a g v e i a p t m E b l r e i n ~ k s t b n 1 8 m o n t h g  
is bigbly resistant to HMRT and setves a an inkdm M r .  ( ~ ~ e t * n B } m k f 5 g r e a I d e m a r a d s o n m e t e s t  
lke determimion of p v k a l  HIY-1 DNA &a several Cmently, the o n t y ~ a s s a y h ~ d d e t e c m o f H W - 1  
predominant in W e r n E umpe a d N orrh 6;s o m  HW-1 
T*e-i-:**-ld3-2-521m834595m38 3:k*ZlW3&SL ~ ~ a n d ~ € m r n h s i a a n d M r l c a a r e n a c ~ b l e ( B r b v r  
-*-~aw- e c ~ i m - e t & # w 1 4 : m a ~ ~ ~  
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a al, 2m; Gwrdin et aL, NM8: Mmmq er al, 2006: Yun eta!, 5 pmol BIO labelled rewrse primer(Eurogente~S. eraing. Belgium). 
Z r n ) .  Forthedetectiono f provirus DNA 100-600 ng(V1aneeLa1,2005) or 
In th i s  context an Ideal PCR-based rrsc sl~ouldb e suf6cientty me isolated DNAand 100copies srandard DNA was used w h ~whas  
sensitive to detect all circulating HIV-1 subtypes and CRFs and taken through the entire sample preparationp rocedure. Amplihca- 
should be applicable wodd wide a t  a price which is affordable in tion of the target fragment was carried out in M R r eaction tubes in 
resource poor counb%esI. t was possible to overcome these pmb- an Eppendorf mastercycler us~ngth e following protocol: thespeci- 
lems by adapting the primers used in  a viral load hst described ficity of the reabion war increased using the Touch Down" (TO) 
prwIously { W e aa l, 2 a ) fo r the arnplificabon and detection PCR (DDn ecaL 1991 ;K h i e a nd Matti& 200&; Zumarmga et at, 
of proviral DNA in human blood samples. 2005). The annealing temperature is s k d egrees above the caicu- 
Iated temperature of the primer pair and decreased over six cycles 
Z Matwials and methods byonedegree/cyde.ARerdenaturationat95 "C for 10 minand inac- 
tivation ofthe UDG,TDtookpEace for six cycIes from 64 "Cdown to 
58 "CA dditional 39 cycles 01 95 'C for 30 s, 5 g  "C for 30 s. 72 "C for 
30aand extensionat 72"C%r4rnin wereused foryeslw answerof 
In addihon to the AIY-I subrypes described in a previous pub- the provirus detechon For the quantifiwtionof KR-produfts only 
lication (Grwe et aS. 20093, the primer system was used for rfie 32 cycles were used a k r i ninal slx I rD cycles. Thisensures that the 
detection of additional subtypes (FljF2. National Institute For Bio- PCR amplification resides in the exponential phase. 
logical Standards and Control (NIBSC) Nr.: ARP 1034; CRF C/A 
N!&SC Nr.: ARP1042: Subtype C, NICBC Nr.: ARP149 and ARP197; 
Subtype H, NIBSC Nr.: ARP 175). 
To control the stability of the pmvirus FCR the fragment of 
interest was amplified our of 200,400,600,800a,n d 1000 ng of im- 
l a mh uman genomlc DNAfroma HIV-l inf-ed patient Thesame 
'Ihe primer design was published elsewhere (CWf%d6 t aL amount of DNA was also amplified from a non ~nfectedp erson to 
ZOM:Drwtenet'al.a006:mfolmesetall,2Wr:WaaL2007; ensure that no nonspecific fragments or satellite bands without rhe 
Soanogj,MO2) and generates a fragment with the sequence of two prwints DNA were generated-The ampl~fiwtionw ar controlled by 
highly consenred regons in the 5' LTR region of the HIV-1 genome. gelelectrophoresis (CyFox Fartec, art i t z  Germany). 
These primers allow only For specific amplification ola single tar- 
get rragment The test described herein 1s based on a three primer 
system (Crwee -t& 2M@j and in this way it enables the amplifi- 
cation of both an internal mnml sequence and the wrrespanding 
provlms sequences. To verify the sensitivity of the PCR system, 1000.500.100.50. 
and 10 copies of the internal Standard were amplified and con- 
23. Entemal conml (DNA HlV- 1 m d o r d )  trolled by gel+lectmphoresis. A standard curve was generated by 
flow cytornetric quantitation of the above listed standard. kch 
The same internal control was used as descbried by Greue eraL copy number was amplified and measured five times. The stan- 
m)ex cept ehe last translation step into RNA. The DNA internal dard curve was used to determine proviral l a d o f 14 proviral DNA 
control was used for the  emaction control of the DNA and for the sarnplesof persons infected w ~ t HhI V. 
PCR 
2.8. PuriJicotion ofthe PCR product 
2.4. HIV-I DNA enrichment and isoEafion 
As recommendedb y the manufacturer. 20 pl of the PCR product 
An aliquotof 200-500 plofthe bled sample wasdituted in2ml was purified with the ornnipure-OLS System (OMN Life Science. 
PBSIBSA and overlaid with 3 rnl lymphme separation med~urn Brernen, Germany) to  remove unused primers. 
(lyrnpho separation medium, MPBiomedicals.OH,USA). Cells were 
separated by centrifugation a t  400 x g  for 20 rnin. The separated 29. Bwds binding mdJlwr ytometry 
lymphxytes were carefully collected and washed by adding 12ml 
PBS and centrifuged again a t  400 x g for 10min. The beads binding p m d u r e a nd the flow cytornetnc rneasure- 
Finally, the  pellet was dissolved in freshly prepared tysis-buffer ment were perfom4 as described previously ( & w ee taL2009). 
(QiAmp DNA Mini WL Qiagen. Germany) and treated as recom- 
mended by rhe manufacturer. 
A standard curve was generated by five independent arnp11- 
In order to avoid inadvertent carryover of fragments ampli- fications and subsequent flow cytometric measurements of the 
fied previously, dWf% instead of dATk were used in any PCR DNA standard. Regression analysis and calculation of the formula 
arnpllficanon and uracil-DNA gtycosylase (UDG)t o digest possi- was done by uslng the Origin 7.5C Software (Ortginbb Cooper- 
ble cross contaminants (PdngetaI, I=). The reaction was carried ation. Northbrnpton. USA). FloMax (Quanrurn Analysis, Munster. 
out a t  37 *C for 15 min before PC!? initiation. The PCR master mix Germany) was used m acquire and process flow cytornetric data. 
included the heollowing components in each 25 jd reaction: 25 pl 
10x buffer: 0.5 pl of the  d m m ix induding 5 mM dWIR. 5 mM 
dlTPs and 10mM dATPs, dGTPs, d m , 0 .5 pI  UDG 1U ivI( Bioron 
Ludwigshafen, Germany); 0.1 @ SuperTaq 5 U/W (HT Biotechol- 3.1. Test specificiFjt 
ogy. UK); 0.1 pl Taq Antibody 1.1 pg(N [Clontech Heidelberg, 
Germany); 5pmol target specifiE DIG labelled forward primer; In add~tions, ubtype C(2arnbia and Tanzania) subtype H (Zaire). 
5 pmol internal mntlol specihc DNP labelled forward primer and CRF A/G and FllR were evaluated (Fig. 1). The internal DNA 
UNIVERSITY OF IBADAN
LIBRARY
-large amw ntofhumanDNA,m r a m p l ~ orae dmon ofrhe spe- Ci6E pmvims ram fragment was visible (& 2). Sample from tndividuak for W wae r u w to  tbe same ampH- - -  lm ' h t m r m a s p e d k ~ o r  
h L m mml--e*-tbe-d- *KBr-s-(RC9 
sysrcmusedlmel: ' I M b p ~ . ~ 2 ~ 3 : ~ ~ ~ Z : ~ W :  
e e w ~ n m : b a c 6 : ~ c z a t m m l a n c f : ~ c f a n e r a i a : ~  
8 : ~ t t a i r e b a c C ~ d  
3.3. P C R m p i g l  
stalsdardWichHW-152TIIspedksequeme~eastlred~~ 
~ d p u r i h c a t i o n a n d-c ation. TO~tbelOWg~dderediou10W.500.1DAM,1Q 
o o p i e s o f t b e ~ s ~ w e r e a m p ~ a n d t h e P a t w a s  
32. PCRsmbiW stoppI33.34atd35 cyd~ahrsixinIiiaIlDqdes.~ampli6- 
cation p d u c t s  wcre visualized by Hdecmphoresis (picture not 
m ~ a s u s e d b o d e t e c t p ~ ( ~ i r a l D N A d W - l a u t 0 s1  b o ~ ~ @ t ~ ~ ~ o n a ~ ~ * 3 9 P C R  
of human m i cD M f mm infeEted Reganihs cyde~(in dud* tibe sbt rD fVcr-) (%3 ). 
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Ibe fragments of the inhrnal mntml and tk dinical smplc5 
were separated by ekxmphoresis on a 2% agamse gel, sbiiml 
with ethidi-de and visualized with a W &ansillumiMbor 
Th-e flow m a m h g p riadplt is stwMn by tk anal- ysis of a pmicive HW-l sample wiih the detection dthe pmvinrs and h ei ntmld ccnh-01 sequ- (Rg4B). Ihe abm m s wee u s d  m test Wood samples of 60 per- sons wi&-  HIV from madan Nisria, 20 pwitive samples finm Germany (n 80) and a0 mn-inf- iodividuals for th ir  
proviral DNA. W e l ~ l m r e s i sa.ra d lbwc ybDmetry showd the 
same ~ I I I O  indicatiag thae depmding on the equipment 
a ~ i h b l e i n a ~.a .m. r y b o t h ~ ba dedd A l l t h e w  s a m p l ~ awerfrae~gaci~wka r-Iprwiral r n e a t h ~ ~ t b a e L a r r r r n t y m c l . m m m ~ d f e f f ~ ~ ~  
DNA Indicating 100% q&Kdydtk m.H IV-1 pmviral DNA of ~ o T ~ b H V - i ~ a ~ ~ ~  
14samples(ea&SPOqg~DNA)wasampdifiedaodquaari- mSpme%bapc"-tglreh-tL)roama& 
wbyflow--1b #W15:CiWW aaLL.1 but in r e u s e . t he overall msb 
n dmmdy high and tfie required labomtwy equipmt is very 
mmp- aod expe&v~ 'hmhre. the published viral load tesr 
wasopt imized(Crrwe~%. ,~~~ampl i fypvi ra lDNAf imm 
~ ~ ~ d p m v i r a l h u r u a u i m m u ~ v h u s T Valpl ~e  H t Y ~ a rCRsFs.dTtse ws&mwas established and 
1 m i r rep eesen& m o f  the m@rd Fawba& tu the tala1 era-  IS S~ECW-&Wie. and reliability e v a h a T~IW  speufidty 
diation of HIV-1. 'Fhe reason of persistwme dHlV inlection in ttae was detennlned by amplIqring ~e same sum from the N l B X  
face ofcurreut -py appears to be mull-' ' ' I and Latent but as reported by ec J. - 
TheuseofaTwch-Domr(lD)KRdescribed a h help ed ~n 
-1 w e r a # n e ~ ~ o f p r i m e r ~ a a d i h e p d u c r Z o a o n  
~ ~ ~ ~ f a 1 4 w ~w ~ c K~R - p m~d w wbh ~ich beawn~e obvioubs in h p -y  & m ~  
M s P m p l c P m r r & t e a d ~ ~ ~ a - m ( s m R  
-L - abgh=dd-mBymmodf8cation.&Istpasitivc si.lple # t w - p r s m ~  t-E   Kr Four of* 80 strnples fmm ttie inktedw hwr that shovwd no W r r J U y  ampliitaon were ideati6sed to twe a DNA mmt (c8n glplr It 1 10J- -  stwddbcQopedabatP6fldtbeisobdWWperP(IRwasused a 4- w w ~ c t 9 . ( 2 m ) h a d ~ m a t ~ ~ r u s D N A L o a d h i g f m e r  3 4- egsdra ma- m than 35I oglOllrg DNA (3162 aopklpg DNA) are rarely evm~ 4 15- 5 =- inpersrmsinFectedwi~~.ThiPmears~rfaeDNAproviral  6 130- - -C-PYlns   load mwimum is abm m €our mpies p r n g DNA In case 7 ZZL - of a proviral laad of QOc copies per pg DNA the deteaion limit 8 9 H-llslWr  -RPIYI*  i sabwt404 lqgDNh~rnaypar t tyexp la in~r rasonfor  lo =- * rmo-d&w-in cbese fwrsamples. 11 n t-m m '13 m- 
14 BJ- m A m-hdw st m amplify pmvItal DNA in samplts d per- 
Km. x a % ~ l r r . d ~ l U f ~ m e m ~b ~ w i t h H t V w i t f i ~ s e m i t i v i t y a n d ~ f i a ~ ~  
UNIVERSITY OF IBADAN LIBRARY
~ + i - ~ u s e t h e s a m t d ~ ~ - l ~ a d  
0nthebasisdtttememodbmiu~kd.aPCR-basedtest 
h t h d ~ 0 f ~ v i 1 d l o a d o f H I V - 2 a a d ~ M i S  
in pmgms. RPC god is  bo measure all m h u tp aramebecs for * 
a s ~ e s s m e ~ t o i H A N I F , t o r e d u d e t t t e c q u i p m e n t r ~ q ~ ~  
t h e m p erkstsub-. 
The aurtrors would Iilte m Annette van Diilmen for -1- 
Lent technical assistam?. 
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