Comparative evaluation of a novel TaqMan real-time reverse transcription–polymerase chain reaction assay for hepatitis A virus detection

Abstract

Objective

To develop and evaluate a novel system for detecting and quantifying hepatitis A virus (HAV) nucleic acid.

Methods

Real-time TaqMan® reverse transcription–polymerase chain reaction (PCR) procedures were established, based on amplification of the highly conserved 5′-non-coding region. Synthetic single-stranded RNA transcripts synthesized in vitro were used as the quantification standard. Ten-fold dilutions were prepared from HAV strain stock suspension to determine precision, accuracy, sensitivity and specificity. In addition, serum specimens from patients with acute HAV underwent clinical evaluation.

Results

The novel assay had a detection limit for HAV RNA of 10 TCID₅₀/ml (where TCID₅₀ is median tissue culture infective dose). It was more sensitive and specific than the commercial quantitative PCR kit manufactured by Shanghai Zhijiang Bio-Tech. However, the Artus HAV RT–PCR kit (Qiagen) had the best performance of the three assays and had a detection limit of 5 TCID₅₀/ml. The new HAV real-time PCR detection system was also successfully applied in 90 serum specimens from patients with confirmed acute HAV infection.

Conclusion

Considering its high reproducibility, sensitivity, specificity and simplicity, this novel amplification system may be suitable for wide clinical application as a diagnostic tool, and for the surveillance and investigation of infectious diseases in developing countries where HAV is endemic.

Keywords

Hepatitis A virus quantification real-time reverse transcription–polymerase chain reaction sensitivity specificity

Introduction

Hepatitis A virus (HAV) is the most common cause of acute hepatitis in developing countries. It is a small, nonenveloped, single-stranded RNA virus that belongs to the genus Hepatovirus in the family Picornaviridae.¹ The HAV genome is composed of a positive-sense single-stranded RNA ∼7.5 kb in length with a 5′-untranslated region (5′-UTR) that is followed by a single open reading frame containing three distinct regions (P1, P2 and P3).² The VP1/P2A junction is the segment of the genome that is used to define genotypes and subgenotypes of HAV strains because this region contains most of the nucleotide variation (> 15%) observed in HAV strains. HAV strains are classified into six genotypes (I–VI), genotypes I–III being of human origin and genotypes IV–VI of simian origin. Each of the human genotypes (I–III) comprises two subgenotypes, A and B. Genotype IA seems to be responsible for most of the HAV infections worldwide.³

Hepatitis A virus infection is a serious threat to public health. There is therefore an urgent need for a widely applicable technique that is able to detect and quantify HAV. Moreover, such a detection technique may be helpful in environmental surveillance, such as the detection of HAV in contaminated water as well as seafood and vegetables that are prone to carrying HAV (such as oysters, mussels, whelks, scallops, cockles, clams, strawberries and tomatoes).^4–8 At present, nucleic acid amplification methods, including conventional polymerase chain reaction (PCR) and real-time PCR assays, play important roles in HAV determination. Nucleic acid amplification assays, with a detection limit (measured as median tissue culture infective dose [TCID₅₀]) of ∼5 TCID₅₀/ml, are considered to be more sensitive than serological assays, and offer many advantages for the rapid and specific detection of viral particles.⁹ In particular, real-time reverse transcription–PCR (real-time RT–PCR) allows quantification of a wide range of viral genome copy numbers in different sample types, and is expected to become widely used in HAV diagnosis and detection. In this study, a novel HAV real-time TaqMan® RT–PCR assay was developed and its specificity and sensitivity were compared with those of two commercial quantitative PCR kits. The effectiveness of the novel assay was further evaluated using clinical samples (serum) from patients with acute hepatitis A infection.

Materials and methods

Ethics

All aspects of the study were performed in accordance with the Chinese national ethics regulations and were approved by the Ethics Committee of the Centre for Disease Control and Protection (CDC), China and by the local CDCs in relevant provinces.

HAV stock and other enteric viruses

The HAV H2-attenuated vaccine strain was purchased from Zhejiang Pukang Biotechnology (Zhejiang, China). Viral stock suspensions contained 3.6 × 10⁶ TCID₅₀/ml, as determined by the vaccine strain supplier. Series of other types of enteric virus were kindly provided by the Poliomyelitis and Diarrhoea Department of the Institute for Viral Control and Prevention (Beijing, China). Two commercial quantitative PCR kits for HAV detection were used for comparison: the RealArt™ HAV LC RT–PCR Kit (Artus, Hilden, Germany) and the HAV Real Time RT–PCR Kit (Zhijiang, Shanghai, China).

Clinical specimens of acute HAV infection

Cases of HAV infection were confirmed in patients on the basis of a positive result in an enzyme-linked immunosorbent assay (ELISA; Kehua Bio-engineering, Shanghai, China) for immunoglobulin (Ig) M antibodies against HAV. Samples used for testing had been obtained, during the 2 years prior to the study, from patients attending Emergency Departments at various medical agencies across China, with verbal patient consent. Briefly, 5 ml of venous blood was collected in a Vacutainer® (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and centrifuged at 2000 g for 10 min at 4 °C. The serum was then isolated and stored at −20 °C until analysis.

Subgenotyping was undertaken, based on phylogenetic analysis of nucleotide sequences in the VP1/2A junction region. Briefly, the nucleotide sequences of the VP1/2A junction region of the HAV strains were obtained by RT–PCR and sequenced in both directions using an automated sequencer (ABI model 373; Applied Biosystems, Foster City, CA, USA). The sequences obtained were edited using the BioEdit Sequence Alignment Editor program (Ibis Biosciences, Carlsbad, CA, USA) and then aligned with published reference strains from different genotypes using the MEGA 3.1 program (Center for Evolutionary Functional Genomics, Tempe, AZ, USA). Neighbour-joining trees were constructed using the Kimura two-parameter correction method. These methods were implemented with software included in the MEGA package.^10,11

Nucleic acid extraction and primer–probe set selection

Genomic viral RNA of the HAV H2-attenuated vaccine strain was extracted from 140 µl of viral stock using a QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was eluted from the QIAamp® spin column in a final volume of 50 µl with RNase-free water and stored at −80 °C until use. To compare analytical sensitivity between the different HAV RNA detection systems, three different sets of 10-fold dilutions (10⁻¹ to 10⁻⁸) were made from the HAV strain stock suspension. RNA extraction was performed on each viral dilution.

To select primer–probe sets, multiple sequence alignments with the HAV sequences available at GenBank (accession numbers X75215, M14707, AF357222, AF512536, AB020568, AB020569, AB258583, AB253604, AY644676, AY644670, D00924) were carried out using Lasergene® 6.0 software (DNASTAR, Madison, WI, USA), and the highly conserved region in the 5′-UTR was subsequently selected for primer and probe design.¹²

RNA standard preparation

An HAV RNA fragment was obtained by in vitro transcription from the linearized plasmid template. Briefly, 5 µg of plasmid was digested with the XbaI restriction enzyme in order to prepare a linearized template. The RiboMAX™ Large Scale RNA Production System (Promega, Madison, WI, USA) was used for full-length transcription of HAV RNA, followed by treatment with RQ1 DNase (Promega, Madison, WI, USA) (1 U/µg cDNA) to remove the DNA template and purification using an RNeasy® Mini Kit (Qiagen). The concentration of prepared RNA was determined using a NanoDrop™ spectrophotometer (ThermoFisher, Wilmington, DE, USA), enabling the number of RNA copies in the solution to be estimated.

TaqMan®-based real-time RT–PCR assay

TaqMan® real-time PCR assays were carried out in a 96-well format using the One Step PrimeScript™ RT–PCR Kit (Takara, Dalian, China). Briefly, 5 µl of extracted RNA was transferred into a capillary containing 20 µl master mix (Premix Ex Taq; Takara, Dalian, China). RT–PCR was performed under optimized conditions in three steps. Reverse transcription was performed at 42 °C for 30 min, followed by denaturation at 95 °C for 10 s. Amplification was achieved by PCR for 40 cycles at 95 °C for 8 s and 60 °C for 34 s. All reactions were performed with an ABI 7500 Real-Time RT–PCR System (Applied Biosystems).

The sensitivity of the reaction was determined by the effective amplification and detection capability of the starting template: the lower the copy number of the starting template, the higher the sensitivity achieved by the system. A variety of enteric viruses and different types of hepatitis virus were chosen for amplification in order to compare the specificity of the new detection method with the specificities of the two commercial kits. These viruses were enterovirus 71, poliovirus, astrovirus, norovirus GI, norovirus GII, rotavirus, adenovirus 40, hepatitis C virus and hepatitis E virus. If no amplification curve was observed and the cycle threshold (C_t) value could not be determined, the system was considered to be highly specific.

Results

Primer–probe set selection

The development of an optimal real-time RT–PCR method relies on the proper selection of target sequences for primers and probe annealing. In this study, HAV sequences available in GenBank, including strains belonging to all human genotypes and subgenotypes so far described, were analysed and the primer–probe set was designed on the basis of highly conserved regions of the HAV genome. The primer–probe set used is shown in Table 1.

Table 1.

Primers and TaqMan® probe used to develop a new real-time reverse transcription–polymerase chain reaction assay for hepatitis A viru.

Position	Primer, probe	Designation	Sequence, 5′–3′
5′-UTR (nt 393–411)	Forward primer	F393	GGT AGG CTA CGG GTG AAA C
5′-UTR (nt 489–508)	Reverse primer	R508	CCT CCG GCG TTG AAT GGT TT
5′-UTR (nt 456–485)	Probe	P5U	FAM-ACA GCG GCG GAT ATT GGT GAG TTG TTA AGA T-BHQ

UTR, untranslated region; nt, nucleotides; FAM, fluorescein amidite; BHQ, Black Hole Quencher™ (Biosearch Technologies, Novato, CA, USA).

RNA standard preparation

One of the crucial steps in real-time quantification is the choice of the most suitable molecule for the generation of the standard curve. In the present study an HAV RNA fragment synthesized in vitro was used to construct the standard curve. The RNA molecule at a concentration of 747.8 ng/µl was calibrated to 10⁷ copies/µl. Serial dilutions (1 : 10) of RNA copy numbers, in a linear range from 10⁷ to 10¹, were used to achieve a reliable standard curve in the subsequent TaqMan® real-time PCR assay. The linear regression equation of the standard curve was y = −3.317 x + 38.228 (R² = 0.999).

Precision and reproducibility analysis

Six virus dilutions were tested. Each dilution was analysed five times by TaqMan® real-time PCR and standard deviations and coefficients of variation (CVs) were calculated to evaluate assay precision. The CV of the new HAV RNA detection system ranged between 4.62% at the dilution of 10⁻¹ and 19.12% at the dilution of 10⁻⁶ (Table 2).

Table 2.

Precision and reproducibility analysis of the quantitative TaqMan® real-time reverse transcription–polymerase chain reaction assay for hepatitis A virus developed in this stud.

Virus dilution	Range of RNA copy number, copies/µl	Mean	Standard deviation	Coefficient of variation, %
10⁻¹	3.52E⁶–3.94E⁶	3.72E⁶	1.72E⁵	4.62
10⁻²	2.24E⁵– 2.92E⁵	2.68E⁵	2.80E⁴	10.45
10⁻³	1.88E⁴–2.45E⁴	2.13E⁴	2.26E³	10.61
10⁻⁴	1.45E³–2.37E³	1.88E³	3.48E²	18.51
10⁻⁵	1.87E²–3.10E²	2.41E²	4.45E¹	18.46
10⁻⁶	1.77E¹–3.43E¹	2.50E¹	4.78E⁰	19.12

Sensitivity analysis

Sensitivity was determined for three independent detection systems: the new in-house TaqMan® real-time RT–PCR assay, the RealArt® HAV LC RT–PCR Kit (Artus assay) and the HAV Real Time RT–PCR Kit (Shanghai Zhijiang assay). These independent experiments were carried out to detect RNA extracted from 10⁻¹ to 10⁻⁸ dilutions of HAV strain stock suspension. All tests were performed in parallel and in quadruplicate on the same dilutions of extracted RNA suspension. The detection limit of the new amplification system was 10 TCID₅₀/ml, which was slightly lower than the 5 TCID₅₀/ml for the Artus assay. However, the TaqMan® real-time RT–PCR assay was ∼10-fold more sensitive than the Shanghai Zhijiang assay. The detection limit of the latter kit was 100 TCID₅₀/mL. Results are shown in Table 3.

Table 3.

Sensitivity of the quantitative TaqMan® real-time reverse transcription–polymerase chain reaction (PCR) assay developed in this study and two commercial quantitative PCR kits, for detection of hepatitis A viru.

	No. of positive samples/no. of tested samples (%)
Virus dilution	Shanghai Zhijiang assay	Artus assay	TaqMan® assay
10⁻¹	4/4 (100)	4/4 (100)	4/4 (100)
10⁻²	4/4 (100)	4/4 (100)	4/4 (100)
10⁻³	4/4 (100)	4/4 (100)	4/4 (100)
10⁻⁴	4/4 (100)	4/4 (100)	4/4 (100)
10⁻⁵	4/4 (100)	4/4 (100)	4/4 (100)
10⁻⁶	1/4 (25)	4/4 (100)	4/4 (100)
10⁻⁷	0/4 (0)	2/4 (50)	0/4 (0)
10⁻⁸	0/4 (0)	0/4 (0)	0/4

Specificity analysis

As expected, none of the other viruses that were tested for gave positive results in either the Artus assay or the TaqMan® real-time RT–PCR assay. However, the Shanghai Zhijiang assay failed to distinguish HAV from enterovirus 71, astrovirus and hepatitis C virus. Results are shown in Table 4.

Table 4.

Specificity of the quantitative TaqMan® real-time reverse transcription–polymerase chain reaction (PCR) assay developed in this study and two commercial quantitative PCR kits for the detection of hepatitis A viru.

Virus type	Shanghai Zhijiang assay	Artus assay	TaqMan® assay
Enterovirus 71	33.16	–	–
Poliovirus	–	–	–
Astrovirus	32.13	–	–
Norovirus GI	–	–	–
Norovirus GII	–	–	–
Rotavirus	–	–	–
Adenovirus 40	–	–	–
Hepatitis C virus	37.59	–	–
Hepatitis E virus	–	–	–

Data are cycle threshold (C_t) values.

–

, C_t value could not be determined.

Assay of clinical samples

The applicability of the new TaqMan® real-time RT–PCR assay to the clinical diagnosis of HAV was validated in serum specimens obtained from 90 patients with confirmed acute HAV infection. As expected, RNA positivity was found for all clinical samples, which included 85 subgenotype IA and five subgenotype IB HAV isolates, and RNA copy number in individual serum samples ranged from 5.18 × 10² to 4.93 × 10⁷ copies/ml when quantified using a synthetic single-stranded RNA standard. The results were 100% consistent with those of the Kehua ELISA test for IgM.

Discussion

The main advantage of real-time PCR assays is the accurate and sensitive determination of starting template copy number over a wide dynamic range. These assays can even detect a single copy of the target gene. Real-time PCR assays can also reliably detect gene copies with lower coefficients of variation than other detection methods. Another advantage of real-time PCR is that data can be evaluated without gel electrophoresis, thereby reducing the time needed for the experiment. Finally, the real-time PCR assay is performed and the data are evaluated in a closed-tube system, thus eliminating the need for postamplification manipulation, and reducing opportunities for contamination. However, the development of an optimal method based on this technique relies on the proper selection of target sequences for primers and probe annealing. Good primer design is particularly important and the design should comply with standard PCR guidelines. There are specific primer design algorithms to minimize the generation of self-complementary primers and interactions between them. Several approaches have been described to circumvent the cosynthesis of nonspecific amplification products in the PCR, including hot-start PCR and, in RT–PCR, the use of a two-step protocol.^13,14 Moreover, primer design to promote the formation of looped structures, in order to suppress the primer dimers or nonspecific amplicon production, has been described and applied with success.¹⁵ However, a two-step strategy in RT–PCR was not used in the present study because we found it offered little advantage over the conventional RT–PCR protocol when HAV nucleic acid was amplified (data not shown).

For HAV RNA detection and quantification, most real-time procedures based on TaqMan® chemistry^16–18 target the highly preserved 5′-UTR region. In contrast, some authors have opted to use primers that target the VP1 capsid¹⁹ or other genomic regions.¹² In the present study, the highly conserved region in the 5′-UTR was chosen when designing the primer pairs and probe targets. This design proved very effective in the subsequent amplification experiments.

Precision and reproducibility tests were performed five times for each dilution of the HAV stock strain and the accuracy of the amplification system was evaluated. Compared with other studies, our primer–probe set showed greater reproducibility, the CV ranging between 4.62 and 19.12%. The CV values obtained using the HAV detection method were much better than those obtained using other primer–probe sets, such as the JN and SH-5U 5′-UTR designs.¹²

Comparison of the analytical sensitivity of the TaqMan® real-time RT–PCR detection system with those of two commercial quantitative PCR kits (in three independent experiments) showed that the Artus test had a slightly better detection limit (5 TCID₅₀/ml) than the new TaqMan® assay (10 TCID₅₀/ml). However, the new TaqMan® amplification system was more sensitive (∼10-fold) than the commercial quantitative Shanghai Zhijiang PCR kit. Furthermore, three independent experiments using serially diluted suspensions of the HAV strain stock solution produced similar quantification data (data not shown). The specificity of these three independent detection systems was tested with several enteric viruses and hepatitis pathogens. Our new real-time RT–PCR assay was similar in specificity to the Artus detection system because no cross-reaction was detected with RNA extracted from poliovirus, astrovirus, norovirus, rotavirus, enterovirus 71, adenovirus 40 and hepatitis C and E viruses in either system. However, positive fluorescence signals were observed with the Shanghai Zhijiang system for astrovirus, enterovirus 71 and hepatitis C virus in the C_t range 32.13–37.59. These positive fluorescence signals may have resulted from probe disruption at the end of the amplification process, in the absence of the target cDNA. On the other hand, nonspecific binding of the primer–probe set to the template is probably another important factor underlying this phenomenon.

The results of this study suggest that our novel TaqMan® real-time RT–PCR assay is more sensitive and specific than some commercial HAV RNA quantification kits. In addition, it was shown to be useful in detecting HAV in clinical samples from patients with a diagnosis of HAV. Thus, this in-house HAV detection method may be helpful in the clinical diagnosis of HAV and may also provide a useful tool for HAV surveillance and investigation.

Footnotes

Acknowledgements

The authors express their sincere thanks to Mrs Qing Zhang and Dr Yong Zhang of the Poliomyelitis and Diarrhoea Department of the Institute for Viral Disease Control and Prevention for kindly providing poliovirus, astrovirus, norovirus, rotavirus, adenovirus 4 and hepatitis C and E viruses.

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by grant from the Major Science and Technology Project for Infectious Disease (2011ZX10004-001) and the 863 Hi-Tech Research and Development Program of China (2011AA02A114).

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