Jpn. J. Infect. Dis., 53, 70‑72, 2000

Short CommumicatiOn

Simultaneous Detection of Hepatitis B, C, and G Viral Genomes

by Multiplex PCR MethodNami Konomil,3, Mari Yamaguchi2, Hideo Naitol, Naoto Aibal ,Takahide Saito3, Yasuyuki Arakawa3 and Kenji Abel *IDepartment of Pathology. National Institute of Infectious Diseases,Toyama 1‑23‑1, Shinjuku Too,0 162‑8640,

2Laboratoyy ofResewch and Development. Sanko Junyaku Co., Ltd.,
Inashiki Ibaraki 300‑1155 and33nd Department ofInternal Medicine, Nihon UniversiO, School of Medicine,

Itabashi Too,0 I 73‑8610, Japan(Received March 7, 2000. Accepted May 9, 2000)
SUMMARY: We established a multiplex polymerase chain reaction (PCR) method for simultaneous detection
ofhepatitis B, C, and G viral genomes. The levels of concordance with the data obtained by conventional single
PCR method were lOO% for slngle infection, 98 to lOO% for double infections, and 92% for triple infections.
This method is not only suited to rapid, large‑scale epidemiologlCal screenlng and clinical diagnosis of thosevirus infections occurrlng alone or in combination, but is also time‑ and cost‑effective.

Multiplex polymerase chain reaction (PCR) is a demanding

for the multiplex PCR for HBV DNA, HCV RNA, and HGVtechnique used for genetic screenlng and other applications

RNA ampliflCations with 300 LLM each dATP, dGTP, dCTP,

where it is necessary to amplifyseyeralproducts simultaneously

and dm, 1.5 mM MgC12,and 40 ng of each primer concentm‑(1‑3). This technique often requlreS extensive optimization
because prlmer‑dimers and other nonspecific products maytion; annealing at 500C for the first round PCR, and employlng
a two‑step annealing temperature at 53oC and 550C for thesecond round PCR. In fact, the multiplex PCR was performed
in a one‑step process that combines CDNA synthesis and PCR
in a slngle tube. That is, for HCV and HGV RNA, the first
PCR was combined with the reverse transc.riptase (RT) step inthe same tube containing 50 LL 1 of a reaction buffer preparedinterferewith the amphfication of specific products (3).
Although prlmer‑dimer formation can usually be avoidedusing hot‑start PCR, amplification specificityis alsoinfluenced
by other factors, such asthe PCR buffer and primer concentra‑tion. At present, PCR is the only reliable method that allowsear)y detection of hepatitis viruSinfections. Further, detectionof hepatitis C vims (HCV) RNA and hepatitis G vims (HGV)
RNA by PCR represents the only means of direct detection

as follows: 1 0 units of RNase inhibitor (Prome芦a, Madisoヮ,Wis･, USA), 100 units ofRT ofMolonFy murlne leukemla
virus (Promega), 40 ng of each outer pnmer for HBV, HCV,of viremia. The current methodology for the screening andand HGV, 300 〃･M of each or the fわur deoxynucleotides, 2detection of those hepatitis viruses in numerous clinicalor in combination, We developed a combined nested PCR

units of AmpliTaq Gold DNA polym.erase (Perkin‑Elmer,Norwalk, Conn., USA), and I X react10n buffer containing
1.5 mM MgC12. To obtain an automatic hot‑start reaction, We
used AmpliTaq Gold DNA polymerase instead of regularthermostable DNA polymerase. The sequences or primers

and multiplex PCR fわr the simultaneous detection or HBV

used for the multiplex PCR were listed in Table 1 along with

DNA and HCV and HGV RNAs in a serum specimen. Ourfindingsindicate that this method is simple, rapid, and highly
sensitive, and could be useful for the screening Ofblood‑bomethe nucleotide position and PCR product size. We previously

samples is inadequate and requlreS numerous PCR reactions
per blood sample. In the present study, to detect hepatitis B

virus (HBV), HCV, and HGV infectionsOccurring either alone

reported the sensitivity of these prlmer combinations in theconventional PCR assay for HBV, HCV, and HGV (4‑6). The
thermocycler was programmed first to incubate the samples

hepatitis virus infections uslng Serum Samples.

Both DNA and RNA were extracted simultaneously fTrom

for 50min at 370C for the initial RT step and then preheat

100 lil of serumusing a nucleic acid extraction kit (SepaGenethem at 950C for 10 min to activate AmpliTaq Gold. This

RV‑良, Sanko Junyaku Co., Ltd., Tokyo), precipitated withwas fわllowed by 40 cycles consistlng Of94oC f♭r 30 S, 50oC

isopropanol, and washed with ethanol. The resulting pellet

for 45 S, and 720C for 1 min uslng a Perkin‑Elmer 2400 or

was resuspended in 50 LEI of RNase‑free water. In order tooptlmize the multiplex PCR, the concentrations or the9700 Thermal Cycler (Perkin‑Elmer). For the second reaction,

deoxynucleotide mixture (100 to 500 〟M), MgC12 (0.5 to 3.0

tube containlng the second set of each inner prlmer,mM), and individual primer pairs (20 to 100 ng) were opti一

deoxymucleotides, AmpliTaq Gold DNA polymerase,and PCR
buffer as in the first reaction, but without RT and omitting

2 LEI (1/25 volume) of the first PCR product was added to amized. After establishing the best condition for the reactlOn,
we further optimized the annealing temperature in the first

the initial 50‑min incubation at 37oC. Amplification was(40 to 55oC)and second round (50 to 55oC) PCR, respectively･
Based on these optlmization results, we designed a protocol

performed for 40 cycles with the followlng Parameters:preheating at 95oC for 10min, 20 cycles ofamplification at
94oC fわr 30 S, annealing at 53oC fわr 45 S, and extension at*Corresponding author: Tel: +8113‑5285‑1111 ext. 2624, Fax:

72oC for lmin, followed by an additional 20 cycles of94oC+8 1‑3‑528511 189, E‑mail: kenjiabe@nih.go.jp

for 30 S, 55oC for45 S, and720C for 1 min. The PCRproducts

70Table 1. Primer sequences used for multiplex PCR
PrimerSequence

Primerpalr

Productsize

For HBV DNA m region)
MD24

5'‑TGCCAACTGGArCCTTCGCGGGACGTCCTT‑3'(nt 1392‑1421)

MD26

5'‑GTTCACGGTGGTCTCCATG‑3'(nt 1625‑1607)

HBx1

5'‑GTCCCCTTCTTCArCTGCCGT‑3'(nt 1487‑1 507)

HBx25'‑ACGTGCAGAGGTGAAGCGAAG‑3'(nt 1604‑1584)

MD24nvm26 (1st)233 bpHBxl/HBx2 (2nd)

1 18 bp

For HCV RNA (5'untranslated region)
I 95 '‑GCGACACTCCACCAmGAT‑3 '(nt 2‑20)

205'‑GCTCATGGTGCACGGTCTA‑3'(nt 330‑3 12)

2 15 '‑CTGTGAGGAACTACTGTCT‑3 '(nt 2846)

225 '‑ACTCGCAAGCACCCTATCA‑3 '(nt 295‑277)

19/20 (lst)329 bp21/22 (2nd)268 bpHGl/HGIR (lst)262 bpHG2/HG2R (2nd)

1 88 bp

For HGV RNA (5'untranslated region)
HG15'‑GGTCGTAAATCCCGGTCACC‑3'(nt I 39‑ 1 58)

HGIR

5㌧CCCACTGGTCCTTGTCAACTl3'(nt 400‑381)

HG25'‑TAGCCACTATAGGTGGGTCT‑3'(nt I 63‑ I 82)

HG2R5'‑jmGAAGGGCGACGTGGACC‑3'(nt 350‑331)

Nucleotide positions deduced &om HBVadr4 (from ref. 8) for HBV, HC‑Jl (from ref. 9) for HCVand HGV‑PNF2161 (&omref. 10) for HGV.

M

1 23456789 1011 12131415

Mー268 bp

M u ltip le又〜 188bp

PCR

ー11Sbpー268bpSingle〜 188bp

PCRーllSbp

Figure. Comparison of results betweenthe multiplex and the conventionalsingle PCR method･were electrophoresed on a 3% agarose gel, stained withslngle PCR assay was shown in Figure. No discrepancy wasethidium bromide, and evaluated under UV light. The sizes

observed between the results of the two assays. To confirmof the PCR products were estimated according to the mlgra‑

the sensitivityand specificity of the PCR, the conventionaltion pattern of a 50‑bp DNA ladder (Pharmacia Biotech,Piscataway, N.J., USA). To confirmthe specificity of theamplification products, sequenclng analysis was performed

uslng 576 serum samples obtained from patients with liver

single PCRand the multiplex PCR were performedinparallel

false‑positive results, PCR assays were done with strict

disease. The results showed lOO% (198/198) concordance insingle infection, 98 to lOO% (90/9 I) in double infections, and
92% (ll/12) in triple infections (Table 2). One case showed

precautions against cross‑contamination. Furthermore, all
PCR assays were performed in duplicate to confirm re‑

HGV RNA was negative by the multiplex PCR. The re‑by the method reported previously (7). To avoid the risk of

discordance of the results in triple infections. In this case,producibilityofthe results bythis PCR method was able to beconfirmed. Furthermore, the specificityof the PCR productsof three different expected sizes in triple infections waspro ducibi lity.

To obtain optlmization of multiplex PCR for HBV, HCV,and HGV, the reaction conditions were assessed by titration

confirmed to correspond to the three genomes by sequence
analysis in 5 cases. No positive reaction was seen in 95

uslng Serial dilutions of chimpanzee serum samples that had

been evaluated forinfectivitywith HBV and HCV, and using
in vitro RNA transcripts for HGV. The sensitivityof this PCR

serum samples from uninfected patients by this assay.

assay allowed detection of as few as 10 copleS Ofthe viral

In the present study, we reported multiplex PCR assay used

genomes ofHBV, HCV, and HGV, respectively; detected byto detect HBV DNA, HCV RNA, and HGV RNA simulta‑

serial dilution assay of the samples. Using this method, We
were able to detect all three viral genomes simultaneouslyin

neously. Evaluation of a serologically known panel of single

5 of 15 serum samples examined in the first experiment.

tion of target sequences by the multiplex PCR method. TheseComparison of ale results between multiplexand conventional

results indicate that our multiplex PCR method could be ainfected or co‑infected individuals showed specific amplifica‑71

Table 2･ Concordance of results betweenthe multiplexand the conventionalsingle PCR method
Single PCR‑positive
Hn=a198

VH

177

CV

HGV

98

B+c
25c+G

B+G

60

ら+C+G

6126

1100%

92%

Multip lex
198

PCR‑positiveConcordancerate

117700%

19800%

12500%

100%

5998%

1

useful tool for large‑scale epidemiologlCal screenlng, and a

5･ Abe, K., Konomi, N. andAbdel‑Hamid, M. (1998): Direct

rapidand cost‑effective method for molecular‑based diagnosis

polymerase chain reaction for detection of hepatitis B, Cof those viral infections.

and G virus genomes from serum without nucleic acidextraction‑simple, rapid and highly sensitive method.

Hepatol. Res., 13, 62‑70.

ACKNOWLEDGMENT

6. Abe, K., Moriyama, M., Hayashi, S., Nakai, K.,

tious Diseases) for his continuous encouragement during thisMiyauchi, I., Edamoto, Y., Saito, T., Fukushima, S.,
Shimizu, T., Matsumura, H. and Arakawa, Y. (1997):

study.

PrevaIence of hepatitis G virus infection among patients

We thank Dr. Takeshi Kurata (National Institute of Infec‑

with liver diseases in Japan. Int. Hepatol. Commun., 6,

This study was supported in part by a grant‑in‑aid for

Science Research from the Ministry of Education, Science,

239‑248.

7. Abe, K. and Konomi, N. (1998): Hepatitis C virus RNASports and Culture orJ叩an and the Ministry orHealth andin dried serum spotted onto rllter paper is stable at room

We胞re of Japan, and by an lntemational Medical Coopera‑temperature. J. Clin. Microbio1., 36, 307013072.
8. Fujiyama, A., Miyanohara, A., Nozaki, C., Yoneyama,T･, Ohtomo, N. and Matsubara, K. (1983): Cloning andtion Research Grant.

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