Journal of Medical Virology 79:1966–1973 (2007)

Comparison of Real-Time Fluorescent RT-PCR and

Conventional RT-PCR for the Detection of Hepatitis
E Virus Genotypes Prevalent in China
Chenyan Zhao,1 Zhuo Li,2 Baoshan Yan,3 Tim J. Harrison,4 Xinhui Guo,2 Feng Zhang,1 Jiming Yin,2
Yan Yan,2 and Youchun Wang1*
1
Department of Cell Biology, National Institute for the Control of Pharmaceutical and Biological Products,
Beijing, PR China
2
Hepatitis Institute, Beijing, PR China
3
Kinghawk Biopharmaceutical Company, Yizhuang, Beijing, PR China
4
Department of Medicine, University College London, London, UK

To compare the specificity and sensitivity of a INTRODUCTION

real-time fluorescent RT-PCR assay with conven-
Hepatitis E virus (HEV) is the causative agent of acute
tional RT-PCR, sera from 110 healthy blood
hepatitis E [Emerson and Purcell, 2003] and is trans-
donors, 120 patients with a clinical diagnosis of
mitted predominantly by the fecal-oral route [Reyes
chronic hepatitis B, and 416 patients with non-A-C
1997]. HEV is a non-enveloped, single-stranded, pos-
acute hepatitis, as well as serial dilutions of HEV
itive-sense RNA virus, and its genome is approximately
genotypes 1 and 4, were tested with both assays.
7.5 kb in size and contains three open reading frames
All samples from healthy blood donors and
(ORFs) [Balayan et al., 1983; Kabrane-Lazizi et al.,
patients with chronic hepatitis B were negative
1999]. Recently, HEV isolates worldwide have been
by both assays. Real-time RT-PCR could detect
classified into four genotypes based on partial or full-
the same final dilution of genotype 1 as conven-
length sequence analyses: genotype 1 (Asian and
tional RT-PCR but could detect a 10-fold lower
African strains), genotype 2 (the prototype Mexican
concentration of genotype 4 than conventional
strain and a few African strains), genotype 3 (strains
RT-PCR. Of 416 samples from patients with a
from sporadic cases in industrialized countries), and
clinical diagnosis of non-A-C acute hepatitis,
genotype 4 (strains from sporadic cases in China, Japan,
127 (30.5%) and 83 (20.0%) were positive for
and Taiwan) [Tam et al., 1991; Huang et al., 1992; Meng
HEV by real-time and conventional RT-PCR,
et al., 1998; Arankalle et al., 1999; Erker et al., 1999;
respectively. The concordance of real-time and
Wang et al., 1999; Schlauder et al., 2000; Wang et al.,
conventional RT-PCR was 80.8%. Furthermore,
2000]. Genotypes 1 and 4 are prevalent in China.
96 and 57 of 171 samples were positive for anti-
During the course of HEV infection, viremia and
HEV IgM by real-time and conventional RT-PCR,
shedding of virus into the feces peak during the
respectively, and 31 and 26 of 245 samples
incubation period and the early acute phase of disease
negative for anti-HEV IgM, were positive by real-
[Balayan et al., 1983; Bradley et al., 1987; Longer et al.,
time and conventional RT-PCR, respectively. All
1993; Tsarev et al., 1993]. HEV RNA can be detected by
amplicons positive by conventional RT-PCR were
RT-PCR in feces and sera from most patients within
sequenced. Of 83 isolates, 7 and 76 belonged to
2 weeks of the onset of illness [Clayson et al., 1995;
genotypes 1 and 4, respectively. Thus, both
Aggarwal et al., 2000]. Prolonged periods of HEV RNA
assays have a high specificity, but the real-time
RT-PCR assay is more sensitive than conven-
tional RT-PCR. Furthermore, HEV genotype 4 is *Correspondence to: Dr. Youchun Wang, MD, PhD, Depart-
ment of Cell Biology, National Institute for the Control of
responsible for most sporadic cases of hepatitis E
Pharmaceutical and Biological Products, No. 2 Tiantanxili,
in the north of China. J. Med. Virol. 79:1966– Beijing 100050, China. E-mail: wychun3@[Link];
1973, 2007. ß 2007 Wiley-Liss, Inc. wangyc@[Link]
Accepted 30 August 2007
KEY WORDS: hepatitis E virus; RNA; real-time
DOI 10.1002/jmv.21040
RT-PCR; genotype; subtype
Published online in Wiley InterScience
([Link])

ß 2007 WILEY-LISS, INC.

Comparison of Two RT-PCRs for HEV RNA 1967

positivity in serum ranging from 4 to 16 weeks also have were collected from 120 patients with a clinical
been reported [Chauhan et al., 1993; Schlauder et al., diagnosis of chronic hepatitis B from the People’s
1993; Nanda et al., 1995]. Moreover, specific antibody Liberation Army 302 Hospital, Beijing, China in 2006
appears during the late incubation period or during and from 110 normal blood donors from Subei Hospital,
the acute phase of illness, as in hepatitis A. Generally, Yangzhou, Jiangsu Province, China in 2006.
anti-HEV immunoglobulin (Ig)M appears first just
before maximal alanine aminotransferase (ALT) activ- Detection of Anti-HEV IgG and IgM Antibodies
ity, reaches a maximum titer around the time of All samples were tested for both anti-HEV IgG and
maximum ALT activity, and disappears about 5 months anti-HEV IgM using an enzyme-linked immunosorbent
into the convalescent phase of the disease [Favorov assay (ELISA) (Genelabs Diagnostics, Singapore, Sin-
et al., 1992]. Anti-HEV IgG first appears shortly after gapore) according to the manufacturer’s instructions.
the IgM response and its titer increases throughout the
acute phase [Dawson et al., 1992; Favorov et al., 1992]. RNA Extraction
Anti-HEV IgG can persist for a long time, as long as
RNA was purified from 140 ml of human serum and a
14 years after infection, although the level decreases
positive control using a QIAamp viral RNA mini kit
rapidly shortly after infection [Bryan et al., 1994;
(Qiagen, Valencia, CA) according to the manufacturer’s
Kabrane-Lazizi et al., 1999].
instructions. The viral RNA was finally eluted from the
The routine diagnosis of hepatitis E is dependent on
spin column in 50 ml of elution buffer and stored at
the detection of antibodies to the virus using enzyme

708C.
immunoassays, particularly HEV-specific IgG and IgM
and, to a lesser extent, IgA. However, antibody assays Real-Time Fluorescent RT-PCR
are not of diagnostic value during the window period of
HEV replication prior to the appearance of specific The HEV ORF3 genomic region (nt 5292–5369,
antibodies [Ahn et al., 2006]. These studies indicate that D11093) is highly conserved based on the alignment of
the detection of specific antibodies to HEV is not full-length HEV sequences from various genotypes
sufficient to diagnose acute HEV infection, especially isolated from humans and swine. Based on the method
in the early stages. The detection of HEV RNA in serum published by Ahn et al. [2006], the primers for the real-
or fecal samples by RT-PCR has been considered the gold time fluorescent RT-PCR were modified to: sense
standard for the diagnosis of HEV infection. However, primer: 50 -CGGTGGTTTCTGGGGTGA-30 and anti-
the treatment of samples for conventional RT-PCR, sense primer: 50 -GCGAAGGGGTTG GTTGGA-30 (Acc
especially nested RT-PCR, is time-consuming and No.D11093, nt 5261–5367); these theoretically are able
expensive, and the nested RT-PCR system is even more to amplify the four genotypes of HEV shown thus far to
prone to cross-contamination and difficult to control infect humans. The probe was the same as the reference
compared to close systems. For this reason, PCR-based paper, namely 50 -FAM-TGATTCTCAGCCCTTCGC-
assays are available only in specialist laboratories and TAMRA-30 [Ahn et al., 2006].
are not commercially available. Recently, real-time The PCR reaction buffer was optimized for MgCl2,
PCR-based fluorescence assays have been developed KCl, and pH using the PCR Optimization Kit (Roche
for the rapid detection of HEV [Mansuy et al., 2004; Orru Diagnostics, Basel, Switzerland). The RT and real-time
et al., 2004; Jothikumar et al., 2005; Ahn et al., 2006; PCR reactions were performed in the same tube. The
Enouf et al., 2006]. Unlike conventional RT-PCR, real- 30 ml reaction mixture consisted of 3 ml of 10 PCR
time RT-PCR is performed entirely within a closed reaction Buffer (containing MgCl2), 0.10 mmol/L of each
system and is not contaminated easily. The procedures primer, 0.20 mmol/L of probe, 2 mmol/L dNTPs, and 10 ml
are also simple and easily standardized. In this study, a of purified RNA. The final reaction contained 2 U of AMV
single-tube real-time fluorescence RT-PCR assay were reverse transcriptase (Promega, Madison, WI) and 4 U
used to detect HEV RNA and its sensitivity and of Taq DNA polymerase (Promega). The thermocycling
specificity were evaluated when used to test clinical conditions were optimized at 30 min at 508C, one cycle of
samples in China. denaturation for 3 min at 958C, followed by five
preliminary amplification cycles of 958C for 10 sec,
MATERIALS AND METHODS 508C for 20 sec, and 728C for 30 sec, then followed by
40 cycles of 958C for 10 sec and 558C for 40 sec.
Serum Samples Amplification, data acquisition, and data analysis were
Sera from 416 patients with a clinical diagnosis of carried out on an ABI PRISM 7000 Sequence Detection
acute sporadic hepatitis were collected from Youan System (Applied Biosystems, Foster city, CA). Negative
Hospital, Beijing, China, during 2005 and 2006. The and non-template controls were included to rule out non-
ratio of males to females was 4.34 to 1. The ages of the specific amplification.
patients were in the range of 18–85 years, while
External Positive Control for Real-Time PCR
the mean and standard deviation (SD) were 49.7 and
13.4, respectively. These sera were negative for anti- Armored RNA containing HEV target RNA was used
hepatitis A virus IgM, anti-hepatitis C virus IgG and as an external positive control. The Armored RNA was
soluble hepatitis B surface antigen. Serum samples prepared as described [Pasloske et al., 1998]. HEV
J. Med. Virol. DOI 10.1002/jmv
1968 Zhao et al.

genotype 1 ORF3 cDNA (D11093 from nt 5261–5367) in (ZCY114), EU107411 (ZCY168), EU107412 (ZCY175),
a recombinant plasmid was digested with Bgl II and Pst EU107413 (ZCY127), EU107414 (ZCY182), EU107415
I and cloned into the pSE380 vector containing a phage (ZCY186), EU107416 (ZCY132), EU107417 (ZCY143),
MS2 gene that can express the capsid protein. When EU107418 (ZCY224), EU107419 (ZCY234), EU107420
the recombinant pSE380 vector was propagated in (ZCY144), EU107421 (ZCY889), EU107422 (ZCY1007),
Escherichia coli DH5a, the recombinant RNA was EU107423 (ZYA1468), EU107424 (ZCY1081), EU107425
synthesized in vivo and packaged into phage particles (ZCY150), EU107426 (ZCY184), EU107427 (ZCY189),
by the capsid protein synthesized from the MS2 gene, EU107428 (ZCY246), EU107429 (ZYAn321), EU107430
forming an Armored RNA. The Armored RNA was (ZYA218), EU107431 (ZCY229), EU107432 (ZCY281),
purified as described [Pasloske et al., 1998]. The EU107433 (ZCY248), EU107434 (ZCY227), EU107435
concentration of the purified Armored RNA particles (ZCY194), EU107436 (ZCY283), EU107437 (ZCY173),
(pseudoviruses) was determined using an extinction EU107438 (ZCY1038), EU107439 (ZCY177), EU107440
coefficient of 0.125 mg/ml and by measuring the (ZCY284), EU107441 (ZCY330), EU107442 (ZCY1099),
absorbance at 260 nm and using the molecular weight EU107443 (ZCY157), EU107444 (ZCY894), EU107445
of the Armored RNA. (ZCY167), EU107446 (ZCY202), EU107447 (ZCY203),
EU107448 (ZCYn471), EU107449 (ZCY1137), EU107450
Conventional RT-PCR (ZCY1143), EU107451 (ZCY1183), EU107452 (ZCY1200),
EU107453 (ZCY1290), EU107454 (ZCY122), EU107455
The oligonucleotide primers for ORF2 used for
(ZCY327), EU107456 (ZCY1119), EU107457 (ZCY1174),
conventional RT-PCR in this study were confirmed to
EU107458 (ZCY90), EU107459 (ZCY130), EU107460
amplify sequences of HEV genotypes 1 and 4 [Wang
(ZCY309), EU107461 (ZCY136), EU107462 (ZCY162),
et al., 1999]. The outer primers, at positions 6298–6321
EU107463 (ZCY1290), EU107464 (ZCY110), EU107465
and 6494–6470 of the Burmese isolate (GenBank
(ZCY1456), EU107466 (ZCY235), EU107467 (ZCY238),
Accession no. M73218), produce a product of 197 bp:
EU107468 (ZCY240), EU107469 (ZCY249), EU107470
ConsORF2-s1, 50 -GACAGAATTRATTTCGTCGGCTGG-
(ZCY251), EU107471 (ZCY100), EU107472 (ZCY247),
30 and ConsORF2-a1, 50 -CTTGTTCRT GYTGGTTRTCA-
EU107473 (ZCY267), EU107474 (ZCY326). The 27 near
TAATC-30 . The inner primers, located at positions 6347–
full-length sequences of HEV genotype 4, used for
6368 and 6491–6467 of the Burmese isolate, yield a
subtype analysis within genotype 4, are as follows:
product of 145 bp: ConsORF2-s2, 50 -GTY GTCTCRG-
HEV subtype 4a (CH-T21, AF151963); 4c (HE-JA28,
CCAATGGCGAGC-30 and ConsORF2-a2, 50 -GTTCRT-
AB220976; HE-JA41, AB220979; HE-JA36, AB220977;
GYTGGTTRTC ATAATCCTG-30 .
JSM-Sap95, AB161717; HE-JA37, AB220978; JKK-
Conventional RT-PCR was carried out in 50 ml
Sap, AB074917; HE-JA19, AB220975; HE-JF5,
reactions with 7 ml RNA, 20 pmol of each primer, 5 U
AB220973; JYW-sap02, AB161719; HE-JF4,
AMV reverse transcriptase, and 5 U Taq polymerase.
AB220972; JTS-SapO2, AB161718; HE-JI4,
Thirty-five cycles of amplification were performed at
AB080575; swJ13-1, AB097811; HE-JF3, AB220971;
948C for 1 min, 508C for 45 sec, and 728C for 1 min, with a
HE-JA1, AB097812; HE-JK4, AB099347; JSN-Sap-FH,
final extension of 728C for 10 min. Second-round
AB091395; JSN-Sap-FH02C, AB200239); 4d (swCH25,
reactions were carried out in a volume of 50 ml with
AY594199; swCH31, DQ450072; T1, AJ272108); 4g
2 ml of first-round product, 20 pmol each primer, and 5 U
(CCC220, AB108537; HE-JA2, AB220974); and other
Taq polymerase. The amplification parameters were the
undesignated subtypes (IND-SW-00-01, AY723745;
same as for the first round.
JAK-Sai, AB074915; HEVN2, AB253420).
Sequence alignments were made using Clustal W to
Sequence Analysis
calculate the evolutionary distances between sequences
All positive amplicons from the conventional RT-PCR and to generate phylogenetic trees. The final output was
were purified and ligated into pMD-18T (Takara, visualized using the program Treeview. The nucleotide
Dalian, China) vectors. One to three clones were identity between sequences was calculated using the
automatically sequenced using vector sequencing pri- program Genedoc 3.2.
mers, an ABI model 373 DNA sequencer, and the ABI
sequencing ready reaction kit (Perkin Elmer, Foster RESULTS
City, CA) according to the manufacturer’s instructions.
Establishment of Real-Time Fluorescent RT-PCR
The sequences determined in this study have been
deposited in the EMBL nucleotide database, accession Real-time RT-PCR was developed using the primers
numbers for the sequences are: EU107392 (ZCY78), and probe designed in this study. Its linearity and
EU107393 (ZCY293), EU107394 (ZCY271), EU107395 sensitivity were evaluated using an HEV pseudovirus
(ZCY198), EU107396 (ZCY171), EU107397 (ZCY193), containing the target HEV RNA sequence. The results of
EU107398 (ZCY153), EU107399 (ZCY283), EU107400 three experiments showed that standard curves were
(ZCY75), EU107401 (ZYA82), EU107402 (ZYA89), generated in the range of 5.6  109 to 5.6  103 copies/
EU107403 (ZCY93), EU107404 (ZYA94), EU107405 reaction using serial 10-fold dilutions of pseudovirus,
(ZYA102), EU107406 (ZCY77), EU107407 (ZCY105), with a detection range from 5.6  1010 to 5.6  103 copies/
EU107408 (ZCY115), EU107409 (ZCY129), EU107410 reaction and a sensitivity of 5.6  103 copies/reaction.
J. Med. Virol. DOI 10.1002/jmv
Comparison of Two RT-PCRs for HEV RNA 1969

The correlation coefficients (R2) were 0.993, 0.993, and remaining 165 samples were negative for both anti-HEV
0.996, and the line slopes were
3.328,
4.499, and IgM and IgG. (Table II).

3.376 for the three experiments.

Comparison of Real-Time RT-PCR With the Comparison of Real-Time RT-PCR With

Conventional RT-PCR for the Detection of Conventional RT-PCR Using Primer Set 1
Serially Diluted Samples of HEV for the Detection of HEV RNA in Sera
Genotypes Prevalent in China From Patients With Acute Hepatitis
To compare the sensitivity of real-time RT-PCR with All 416-serum samples from acute hepatitis cases
conventional RT-PCR for the detection of HEV geno- were tested for HEV RNA using both real-time RT-PCR
types prevalent in China, genotypes 1 and 4 were and conventional RT-PCR with primer set 1. If the
serially diluted 10-fold. All dilutions were tested results of the two assays were discrepant, the samples
three times by real-time RT-PCR and the conventional were run duplicate using each of assays. The sample was
RT-PCR. Real-time RT-PCR had the same sensitivity as considered as positive only if two of the three
conventional RT-PCR when detecting genotype 1, but results were positive. Of the 416 samples, 127 (30.5%)
was 10 times more sensitive than conventional RT-PCR and 83 (20.0%) were positive by real-time RT-PCR and
in detecting genotype 4 (Table I). conventional RT-PCR, respectively. The concordance of
real-time RT-PCR and conventional RT-PCR was 80.8%,
with 65 samples classified as positive and 271 samples
Assay Specificity classified as negative by the two assays. Specifically, five
of eight samples positive for only anti-HEV IgM were
We tested the real-time RT-PCR assay on a total of
positive by real-time RT-PCR, while two of eight
230 serum samples from individuals who were not
samples were positive by conventional RT-PCR, giving
suspected to be positive for HEV, including 110 samples
a concordance rate of 62.5%. Of 163 samples positive for
from normal blood donors and 120 samples from
both anti-HEV IgM and IgG, the concordance of the two
patients with a clinical diagnosis of chronic hepatitis
assays was 66.9%, and the positivity rates for real-time
B. Eight of the 230 serum samples were positive for anti-
RT-PCR and conventional RT-PCR were 55.8% and
HEV IgM and 16 were positive for anti-HEV IgG. The
33.7%, respectively. Of the 80 samples that were positive
specificity of the real-time RT-PCR assay was evaluated
for only anti-HEV IgG, the concordance rate was 81.3%
by testing HEV RNA from 230 serum samples that were
and the positive rates were 25.0% and 18.8%, respec-
negative for HEV RNA using the conventional RT-PCR
tively. For the 165 samples negative for both anti-HEV
assay with primer set 1. All 230-serum samples were
IgM and IgG, the concordance rate was 95.2% (Table II).
negative for HEV RNA by the real-time RT-PCR assay.
The positive rates in the four populations were signifi-
Therefore, the specificity of the real-time RT-PCR assay
cantly different (P < 0.05) between the two assays.
in this study was 100%.
Among 127 samples that were positive by real-time
RT-PCR, 91 had threshold cycle (Ct) values below
28.0 and the remaining 36 samples had Ct values
Detection of Anti-HEV IgG and IgM
greater than 28. The lower the Ct value, the higher the
Antibodies by ELISA
presumptive viral load. Of the 91 samples with a higher
All 416 samples from acute sporadic hepatitis cases HEV viral load, 60 were positive by conventional RT-
were tested for anti-HEV IgG and IgM. Of these, PCR. The concordance of the two assays was 65.9% in
245 were positive for anti-HEV IgG and 171 were this group. When the Ct values were between 28 and 34,
positive for anti-HEV IgM. Therefore, the positivity 5 of 36 samples were positive by conventional RT-PCR,
rates for anti-HEV IgG and IgM were 58.9% and 41.1%, and the concordance of the two assays was 13.9%. These
respectively. Eight samples were positive only for anti- data indicated that most samples with a lower HEV viral
HEV IgM, 163 were positive for both anti-HEV IgM and load could be detected by real-time RT-PCR rather than
IgG, and 80 were positive for only anti-HEV IgG. The conventional RT-PCR.

TABLE I. Comparison of Real-Time RT-PCR With Conventional RT-PCR for the Detection of HEV RNA in
Serially-Diluted HEV Sera

Dilution
0 1 2
Methods Genotypes 10 10 10 103 104 105
Real-time Gen 1 þ þ þ þ þ

RT-PCR Gen 4 þ þ þ þ þ

Conventional
RT-PCR Gen 1 þ þ þ þ þ

Primer set 1 Gen 4 þ þ þ þ


Note: All dilutions were tested in triplicate; ‘‘þ’’ indicates that all three tests were positive.

J. Med. Virol. DOI 10.1002/jmv

1970 Zhao et al.

TABLE II. Comparison of Real-Time RT-PCR With Conventional RT-PCR for the Detection of HEV RNA

Real-time Real-time PCR Conventional Conventional RT-PCR Concordance of

NO. PCR positive rate (%) RT-PCR positive rate (%) two PCR (%)
IgMþIgG
8 5 62.5 2 25.0 62.5
IgMþIgGþ 163 91 55.8 55 33.7 66.9
IgM-IgGþ 80 20 25.0 15 18.8 81.3
IgM-IgG
165 11 6.7 11 6.7 95.2
Total 416 127 30.5 83 20.0 80.8

Analysis of HEV Sequences Isolated China. The results indicated that different primer sets
From Clinical Serum Samples had different sensitivities for the detection of isolates
from various genotype. Primer set 1, which had the
Eighty-three products from conventional RT-PCR
highest sensitivity for detecting each isolate, was
were cloned and sequenced. The length of the amplicons
selected for this study.
was 145 nucleotides and the novel sequences were
Real-time nucleic acid amplification systems, which
98 nucleotides in length after removal of both primer
are reliable tools with a high sensitivity and specificity
sequences; these were compared with the corresponding
and fast turnaround times, have recently been devel-
regions of HEV genotypes 1, 2, 3, and 4 reported
oped. Two dominant real-time RT-PCR methods
previously. Eight sequences (ZCY78, ZCY153, ZCY171,
reported to detect HEV are a SYBR Green RT-PCR
ZCY193, ZCY198, ZCY271, ZYA286, ZCY293) were 92–
assay and a TaqMan assay. The SYBR Green RT-PCR
100% identical to HEV genotype 1 and appear to belong
[Orru et al., 2004] is a one-step assay that uses shorter
to HEV genotype 1. The other 75 sequences were 73–
primers (15–16 mer) than those typically used in RT-
84%, 77–86%, 75–84%, and 84–95% identical to HEV
PCR and PCR assays. The short primers raise the
genotypes 1, 2, 3, and 4, respectively, and appear to
potential for detection problems due to the low Tm of the
belong to HEV genotype 4. They share 76–100% identity
primers and the possibility of non-specific amplification
with each other at the nucleotide level. The 75 sequences
of non-target nucleic acid. The TaqMan assay [Mansuy
could be clearly grouped into three or more clades, one of
et al., 2004] is a two-step RT-PCR assay that separates
which includes 30 isolates (ZCY1143, ZCY240, ZCY143,
the RT step from the PCR step, which increases
ZCY184, ZCY1290, ZCY1099, ZCY233, ZCY248,
handling and the possibility of contamination. Perform-
ZCY283, ZCY1137, ZCY238, ZCY1200, ZCY251,
ing both RT and PCR in a single tube is preferable,
ZCY132, ZCY284, ZCY130, ZCY77, ZCY127, ZCY75,
because it reduces handling and therefore minimizes
ZCY105, ZCY189, ZCY330, ZCY150, ZCY162, ZCY1183,
the chance of pipetting errors and contamination.
ZCY249, ZCY246, ZCY114, ZCY144, ZCY136) sharing
The primers and probe used in this study are highly
82–96% identity with T21, which is the representative
specific for HEV sequences of all four genotypes.
isolate of subtype 4a, and formed a branch with T21.
However, the TaqMan assay reported by Mansuy et al.
Thus, they could belong to subtype 4a. The second clade,
[2004] uses degenerate primers and a degenerate probe,
including 20 isolates (ZCY1081, ZYA218, ZCY115,
which may reduce the sensitivity and specificity of the
ZCY175, ZCY229, ZCY889, ZYA82, ZCY182, ZCY234,
assay when used to detect heterogeneous targets. By
ZYA89, ZYA94, ZYA102, ZCY129, ZYA1468, ZCY1007,
optimizing the reaction conditions (including concen-
ZCY224, ZCY93, ZCY168, ZCY186, ZYAn321), was 84–
trations of primers, probe, Taq DNA polymerase, and
93% identical to T1, which was the representative
AMV reverse transcriptase, the temperature of reverse
isolate of HEV subtype 4d, and formed a branch with
transcription, and PCR cycling parameters), a rapid and
T1. Therefore, the second clade could belong to subtype
sensitive diagnostic assay was designed for the direct
4d. The remaining sequences were very variable and
detection of HEV RNA in serum samples by performing
shared 83–100% identity with each other. They did not
the RT and PCR reactions in one tube. The samples were
from a branch with subtypes 4c or 4g and therefore did
classified as positive or negative based on the Ct values
not belong to either subtype (Fig. 1).
of sera, because the Ct represents the starting template
copy number even if it does not provide the quantitative
DISCUSSION
copy number. By using a serial dilution of psuedovirus
Since the detection of HEV antibodies is limited to a containing the HEV target sequence, the detection limit
small window period during HEV infection, RT-PCR and linearity of the real-time RT-PCR ranged from
assays, including nested RT-PCR, for HEV RNA have 5.6  1010 copies/reaction to 5.6  103 copies/reaction
been developed in different specialist laboratories. and from 5.6  109 copies/reaction to 5.6  103 copies/
However, different laboratories may use different sets reaction, respectively. To date, no HEV RNA standard is
of primers. Thus the sensitivity of those RT-PCRs, available. The sensitivities of other published assays
especially when detecting different genotypes, may vary have been expressed in various ways and are difficult to
with the primers. In this study, the sensitivity of compare. The real-time RT-PCR described by Enouf
conventional RT-PCR for detecting HEV with different et al. [2006] can detect 10 copies of HEV (DNA) plasmid
primer sets was evaluated using genotypes prevalent in but the assay of Li and Krawczynski [2006] can only
J. Med. Virol. DOI 10.1002/jmv
Comparison of Two RT-PCRs for HEV RNA 1971

Fig. 1. Phylogenetic analysis of HEV genotype 4 isolates based on 98 nucleotides from ORF2. Scale bar
indicates 0.1 nucleotide substitutions per position.

detect 4.5  103 copies of synthetic HEV RNA standard real-time RT-PCR is significantly higher that that
per reaction, while the assay in this study can detect detected by conventional RT-PCR. Thus the sensitivity
5.6  103 copies of pseudovirus (RNA) per reaction. This of real-time RT-PCR is higher than that of conventional
assay also was evaluated by analyzing simultaneously RT-PCR. Meanwhile, some samples from acute hepatitis
clinical samples, using a more sensitive conventional cases, which were negative for both anti-HEV IgM and
RT-PCR. In samples negative for HEV RNA or anti-HEV IgG, were positive for HEV RNA by both assays. Those
antibody, no HEV RNA was detectable by real-time RT- samples were likely in the window period of HEV
PCR. Thus, its specificity approaches 100% for those infection, when specific antibodies had not yet been
samples. Of the 416 serum samples from sporadic acute produced. Therefore, the detection of HEV RNA may
hepatitis cases that were positive for anti-HEV IgM, 171 facilitate early diagnosis of HEV infection.
could be diagnosed as positive for hepatitis E. Of these, Although the assay could not be used to quantify the
96 and 57 were positive for HEV RNA by real-time RT- amounts of virus, the level or titer of viral load could be
PCR and conventional RT-PCR, respectively. The con- estimated based on the Ct value. The lower the Ct value,
cordance between anti-HEV IgM and RNA detected by the higher the presumptive viral load. Among 91
J. Med. Virol. DOI 10.1002/jmv
1972 Zhao et al.

samples with Ct values below 28.0, which indicated a The pathogenicity of HEV genotypes 3 and 4 for
higher viral load, approximately 66% (60/91) were humans is low and symptomatic disease rarely is
positive by conventional RT-PCR, while only 14% (5/ observed [Emerson and Purcell, 2003]. However, when
36) of samples with Ct values less than 28.0 were we analyzed clinical data from 36 patients infected with
positive. In general, this result suggests real-time RT- HEV genotype 4, we found that the concentration of total
PCR is more sensitive than conventional RT-PCR. bilirubin (TBIL) and ALT could reach to 240.5 mg/dl and
However, the real-time RT-PCR still missed some 2568 U/L, respectively. These data indicate that HEV
samples which were positive using the conventional genotype 4 may cause severe liver damage. Thus, as
RT-PCR. Therefore, the sensitivity still needs to be more and more strains of HEV genotype 4 are isolated
improved. from around the world, its pathogenicity is worth
In this study, all the results of real-time RT-PCR were further study.
determined by cycle time (CT) and the fluorescent signal
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