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Rapid detection of the hepatitis B virus YMDD mutant using AllGlo™ probes
Clinica Chimica Acta 412 (2011) 1018–1021
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Rapid detection of the hepatitis B virus YMDD mutant using AllGlo™ probes Feng Zhao-lei a, Yu Xiao-yan b, Lu Zhi-ming c, Geng Da-ying a, Zhang Li a, Chen Shi-jun a,⁎ a b c
Department of Center Laboratory, Jinan Infectious Disease Hospital, Shandong University, Jinan, Shandong 250021, PR China Department of Laboratory Medicine, Jinan central Hospital, Jinan, Shandong 250014, PR China Department of Laboratory Medicine, Shandong Provincial Hospital, Shandong University, Jinan, Shandong 250021, PR China
a r t i c l e
i n f o
Article history: Received 31 August 2010 Received in revised form 27 January 2011 Accepted 8 February 2011 Available online 12 February 2011 Keywords: Hepatitis B virus YMDD mutant Real-time PCR AllGlo™ probe
a b s t r a c t Background: The early detection of hepatitis B virus (HBV) mutants in clinical samples is important when monitoring chronic HBV patients with lamivudine-resistant mutations during lamivudine therapy. Methods: The AllGlo™ probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The sensitivity and specificity of the assay were evaluated using a series of diluted mixtures of wild-type and mutant plasmids. This assay was compared with direct sequencing and the mutation-specific primer assay. Results: Each YMDD, YVDD, and YIDD probe only detected its corresponding plasmid. Moreover, the assay correctly identified negative samples from 40 non-HBV infected patients and 100 healthy controls. The detection limit of this assay was 50 copies/ml for YVDD and YIDD. The assay could detect the mutant strains when they were present at ≥ 10% within a mixed virus population. The assay was fully concordant with direct sequencing in 34 samples (56.7%) and partially concordant in 26 samples (43.3%), and detected more types of the HBV motif than direct sequencing. Conclusions: AllGlo™ probe assay is a novel, sensitive and specific assay to detect lamivudine-related HBV mutants, therefore, may be useful for monitoring chronic HBV patients treated with lamivudine. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a major health problem worldwide with considerable morbidity and mortality. Currently it is estimated that N2 billion people have been infected with HBV, and 300 million people have chronic HBV infection [1]. HBV is one of the leading causes of chronic liver disease, including liver cirrhosis, hepatic failure and hepatocellular carcinoma (HCC), especially in developing countries [2]. Nucleoside analogue therapy allows safe, long-term suppression of HBV and is a major milestone in the treatment of chronic hepatitis B. Lamivudine, the first-line antiviral agent approved by FDA, can effectively suppress HBV replication, reduce disease activity, improve liver histology, and delay clinical progression [3–5]. However, long-term monotherapy is usually associated with the emergence of resistant mutants, followed by increases in viral load and reelevation of aminotransferase activities. Genotypic resistance could be detected in 14% to 32% of patients after 1 year of treatment [3,6,7]. In Asia, genotypic resistance increased from 14% in year 1 to 38%, 49%, 66%, and 69% after 2–5 years of treatment, respectively [8–10]. Most lamivudine-resistant
⁎ Corresponding author at: Department of Center Laboratory, Jinan Infectious Disease Hospital, Shandong University, 173 Jingshi Rd, Jinan, Shandong 250021, PR China. Tel.: + 86 531 87924945; fax: + 86 531 87069102. E-mail address: [email protected] (S. Chen). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.012
strains are associated with mutations of the YMDD motif in the C domain of HBV polymerase. The key mutations are the substitutions of methionine at the rtM204 with either isoleucine (rtM204I, YIDD variant) or valine (rtM204V, YVDD variant) [11,12]. Several assays including fragment length polymorphism (RFLP) [13], the Line Probe Assay (INNOLiPA, Innogenetics Inc, Belgium) [14], peptide nucleic acid clamping [15], mass spectrometric analysis [16], fluorescence polarization [17], microarray [18,19], reverse dot blot [20], denature gradient gel electrophoresis [21], sequencing [22], fluorescence quantitative PCR [23], real-time PCR using mutation-specific primers [24], TaqMan probes [25], or TaqMan-minor groove binder probes [26] have been used to rapidly detect and quantify lamivudine-resistant mutations, but the sensitivity and accuracy of these methods should be further improved. In this study, we established a novel rapid, sensitive, and accurate assay based on real-time PCR using AllGlo™ probes [27] for detecting the YMDD mutations within HBV. The AllGlo™ probe has 2 identical reporter dyes that quench each other and become de-quenched once the probe is cleaved. Upon hybridization with the target sequence, the labeled oligo becomes stretched and cleaved. This leads to the separation of the two reporter dyes and consequently de-quenching occurs. Because this process releases two fluorescing molecules instead of one, the signal change is much greater, making AllGlobased quantitative PCR detections highly sensitive and robust. Using AllGlo™ probes allowed us to rapidly detect HBV mutants in clinical
Z. Feng et al. / Clinica Chimica Acta 412 (2011) 1018–1021
samples with high sensitivity and accuracy. This method may be useful for monitoring lamivudine-resistant mutations during lamivudine therapy. 2. Materials and methods 2.1. Patients and HBV DNA extraction Serum samples were collected from 60 chronic hepatitis B patients who had received lamivudine monotherapy for at least 6 months and were later found to have increased viral titers. All specimens were stored at −70 °C until tested. The levels of HBV DNA (5.67± 1.31 log copies/ ml) were measured by real-time PCR (PG Biotech, China) in an ABI-7500 thermal cycler (Applied Biosystems, Foster City, CA). Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of our hospital. HBV DNA was extracted (200 μl) in serum samples using the QIAamp blood kit (Qiagen, Hilden, Germany) and following the manufacturer's protocol. 2.2. External standards To validate the specificity and sensitivity of the assay, three external standards were constructed for the three type-specific reactions (wildtype: 204M [ATG], mutant: 204V [GTG], and mutant: 204I [ATT]). Briefly, sera with wild-type HBV DNA, YVDD, or YIDD mutants were amplified by PCR with primer sets (Table 1). The obtained PCR products were then cloned into pMD19-T Simple Vector (TakaRa, Dalian, China) using the standard protocol. Each plasmid was verified through sequencing and linearized by Sac I digestion. The copy numbers were then assessed by absorption spectrophotometry and adjusted to 1 × 101, 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 copies/ml. 2.3. Real-time PCR using AllGlo™ probes The primers and probes (Chaoshi BioTechnologies, Shanghai, China) used in this study are listed in Table 2 [26]. Three separate reaction mixtures were performed to detect wild-type YMDD, YIDD mutant, or YVDD mutant, respectively. PCR amplification was performed in a 96well plate in a 25 μl volume containing 5 μl of the extracted HBV DNA sample (or external standard), 0.4 μmol/l of a forward primer (Table 2), 0.4 μmol/l of a reverse primer, 0.2 μmol/l of a type-specific probe (YMDD and YVDD, or YIDD), and 12.5 μl of Universal PCR Master Mix (Applied Biosystems). Real-time PCR was performed on a 7500 Real-time PCR system (Applied Biosystems) and analyzed using 7500 system SDS software. All of the reactions were performed three times. The programs used were as follows: 50 °C for 1 min and 95 °C for 1 min for the initial period, and then 95 °C for 15 s followed by 62 °C for 1 min for 40 cycles. 2.4. Real-time PCR using mutation-specific primers and TaqMan probe Real-time PCR using mutation-specific primers was performed as described previously [24]. In brief, parallel reactions C, V, and I corresponding to the individual's reverse primer (Table 3) were used to detect total HBV YMDD, YVDD, and YIDD respectively.
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Table 2 Primers and probes for detection of wild-type YMDD, YVDD and YIDD mutants. Primers or probes
Sequence
Y(M/I/V)DD-F2 Y(M/I/V)DD-R2 YMDD-probe YIDD-probe YVDD-probe
5′-GGGCTTTCCCCCACTGTT-3′ 5′-GTACAGACTTGGCCCCCAATAC-3′ 5′-AllGlo-CTTTCAGTTATATGGATGATG-AllGlo-3′ 5′-AllGlo-CTTTCAGTTATATTGATGATGTG-AllGlo-3′ 5′-AllGlo-CTTTCAGTTATGTGGATGAT-AllGlo-3′
PCR was performed using 5′-TGGGCCTCAGTCCGTTTCTC-3′(HBV nt647– 666) and 5′-GGACTCAAGATGTTGTACAG-3′ (HBV nt786–767) as the sense and antisense primers, respectively, and sequenced by ABI 3100 Analyser (Applied Biosystems). 2.6. Subcloning of the HBV gene Of the samples whose detected results were partially concordant between direct sequencing and real-time PCR using AllGlo™ probes, 10 samples were selected at random for PCR amplification. The PCR products were then cloned into the pMD19-T vector and sequenced, as described by Kobayashi et al. [29]. 3. Results 3.1. Specificity of the assay Cross-reactivity tests were performed to study the specificity of the assay based on real-time PCR using AllGlo™ probes. A diluted series of each plasmid (ranging from 10 × 108 to 10× 101 copies/ml) was used as the template for PCR using AllGlo™ probes. A negative control was included to avoid nonspecific amplification. Each single YMDD, YVDD, and YIDD probe was able to only detect its corresponding plasmid. Plasmid mixtures containing the wild-type (YMDD) and mutants (YVDD and/or YIDD) between 1 × 102 and 1 × 108 copies/ml could be detected only by their corresponding probes. Also, when the assay was used to detect serum samples from 40 non-HBV infected patients and 100 healthy controls, negative results were obtained, implying the specificity and accuracy of this assay (data not shown). 3.2. Sensitivity and detection limits of the assay Mixing experiments were performed to determine the sensitivity of each probe in the assay. The YMDD plasmids and mutant plasmids (YIDD or YVDD) were mixed at ratios of 100:0, 90:10, 80:20, 50:50, 20:80, 10:90, and 0:100, corresponding to a final concentration of 108, 107, 106, 105, 104, 103, 102, and 10 copies/ml, respectively. Then the three plasmids (YMDD, YIDD, and YVDD) were mixed at ratios of 90:5:5, 80:10:10, 50:25:25, 20:40:40, 10:45:45, and 0:50:50, corresponding to the same concentration of 108, 107, 106, 105, 104, 103, 102, and 10 copies/ ml, respectively. Among the 500 copies/ml present in the mixtures, 10% of the mutants – either YVDD or YIDD – were detected. This finding demonstrates that the detection limit of the assay was 50 copies/ml.
2.5. Direct sequencing Direct sequencing of the DNA sequence of domain C of the HBV polymerase gene was carried out as described previously [28]. Briefly, Table 1 Primers for wild-type HBV DNA, YVDD and YIDD mutation amplification. Primers
Sequences
Y(M/I/V)DD-F1 Y(M/I/V)DD-R1
5′-GATGTGTCTGCGGCGTTTTA-3′ 5′-CAGCAAAGCCCAAAAGACCCAC-3′
Table 3 Probe and mutation-specific primers for YMDD, YIDD and YVDD. Denomination
Sequences
Probe Forward primer Reverse primer C Reverse primer V Reverse primer I
5′-FAM-AGCCCTACGAACCACTGAAC-TAMRA-3′ 5′-CCTATGGGAGTGGGCCTC-3′ 5′-GCCCCCAATACCACATCATC-3′ 5′-CCCAATACCACATCATCCAC-3′ 5′-CCCCCAATACCACATCATCA-3′ 5′-CCCCCAATACCACATCATCG-3′ 5′-CCCCCAATACCACATCATCT-3′
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Z. Feng et al. / Clinica Chimica Acta 412 (2011) 1018–1021
3.3. Comparison of direct sequencing with real-time PCR using AllGlo™ probes or mutation-specific primers Three methods were compared: direct sequencing, real-time PCR using AllGlo™ probes (AllGlo™ probe assay), and real-time PCR using mutation-specific primers (mutation-specific primer assay). The results of these three methods performed on 60 serum samples from chronic hepatitis B patients receiving lamivudine monotherapy were analyzed as described by Evelien Libbrecht [30]. Each assay result was divided into four classes: fully concordant, partially concordant, partially discordant, and completely discordant with direct sequencing. Results were considered fully concordant if both direct sequencing and the AllGlo™ probe assay or the mutation-specific primer assay showed the same results. The results were considered partially concordant if the AllGlo™ probe assay or the mutation-specific primer assay provided additional information compared to that provided by direct sequencing. For example, the AllGlo™ probe assay and the mutation-specific primer assay showed a mixture of wild-type and mutant sequences, while direct sequencing showed only a wild-type or a mutant sequence. The results were considered partially discordant if direct sequencing showed a mixture of wild-type and mutant sequences, while the AllGlo™ probe assay and the mutation-specific primer assay showed a wild-type sequence or a mutant sequence only. The results were considered completely discordant if one test showed a mutant and the other test resulted in a wild type. As demonstrated in Table 4, the results produced by the AllGlo™ probe assay and direct sequencing were fully concordant in 34 samples (56.7%) whether it was a single-type or a mixed-type. The results were partially concordant in 26 samples (43.3%). In all partially concordant samples, the AllGlo™ probe assay detected more types of motifs (YMDD, YVDD, or YIDD) than that provided by direct sequencing, meaning that our assay provided more information. Of these 26 samples, 10 samples were selected at random to amplify and subclone. Then 20 randomly selected clones per sample were picked and sequenced. The results produced by subcloning were fully concordant with that produced by our AllGlo™ probe assay. The results produced by the mutation-specific primer assay and direct sequencing were fully concordant in 47 samples (78.3%), partially concordant in 3 samples (5.0%), partially discordant in 7 samples (11.7%), and completely discordant in 3 samples (5.0%), respectively. The results produced by the three methods were fully concordant in 27 samples (45.0%) (Table 4). 4. Discussion While new and effective nucleotide/nucleoside analogues are increasingly available for the treatment of chronic HBV infection, for reasons of efficacy, safety, and cost, lamivudine remains the first-line therapy in developing countries with a high incidence of chronic HBV infection. However, the beneficial effects of antiviral agents in halting disease progression are blunted by the occurrence of drug mutations. Severe hepatitis reactivation due to drug-resistant virus can result in hepatic decompensation and even mortality. Such unsatisfactory out-
Table 4 Concordant results obtained through blind analysis using three methods of 60 serum samples. Type
Sequencing vs. AllGlo™ probe assay
Sequencing vs. mutation-specific primer assay
Fully concordant Partially concordant Partially discordant Completely discordant Total
34 26
47 3 7 3
60
60
comes could be prevented through early detection, thereby allowing clinicians to select appropriate alternatives. In this study, we described a real time PCR based on AllGlo™ probes for genotyping the YMDD motif variants in clinical samples from HBVinfected individuals. This new approach has several potential advantages over other methods. First, this assay is more sensitive than direct sequencing or a mutation-specific primer assay. The detection limit of the real time PCR based on AllGlo™ probes is 50 copies/ml for YVDD and YIDD with 10% sensitivity within a mixed virus population. This assay also has high specificity. It could detect YMDD mutants at a copy number ranging from 50 to 108. Due to its wide detection range, this assay can be applied in a clinical context for detection of YMDD mutants having a low amplification efficiency. Hua et al. [26] reported a novel, rapid, sensitive, and accurate method for detecting YMDD mutants using TaqMan-minor groove binder probes (MGB) probes, which have a fluorophore at the 5′-end and a nonfluorescent quencher at the 3′-end. These probes can produce very low background and strong hybridization-triggered fluorescence. This method's detection limit is 10 copies for YVDD and 50 copies/ml for YIDD with 10% sensitivity within a mixed virus population. Using the AllGlo™ probes, the detection limit of our assay is 50 copies/ml for both YVDD and YIDD. The AllGlo™-based quantitative PCR detections are highly sensitive and theoretically robust because the AllGlo™ probe has two identical reporter dyes that quench each other and become dequenched once the probe is cleaved. When de-quenching occurs, two fluorescing molecules are released, instead of one. This creates a much greater signal change. Therefore, the detection limit of our assay is theoretically more sensitive than that of the method using TaqManMGB probes. We inferred that the lower sensitivity of our assay might be associated with the quality and/or quantity of nucleotides extracted from plasmids. To compare the concordance of direct sequencing with the AllGlo™ assay or the mutation-specific primer assay, 60 clinical samples were analyzed using three methods: direct sequencing, AllGlo™ probe assay, and mutation-specific primer assay. Fully concordant results of the AllGlo™ probe assay with direct sequencing were observed in 34 samples (56.7%). Partially concordant results were observed in 26 samples. Neither partially discordant nor completely discordant results were observed. Moreover, there were more genotypes detected by this assay than by direct sequencing in the partially concordant samples. Results from subcloning experiments conducted on 10 randomly selected samples were consistent with this assay, suggesting the accuracy and superiority of our novel method. Although direct sequencing had previously been considered the “gold standard” due to its accuracy, its sensitivity is limited (approximately 20% of the total virus population) [26]. The ability to detect only partially mixed genotypes might be associated with its relatively low sensitivity. The mutation-specific primer assay is also a sensitive method for detecting YMDD mutants. Although the fully concordant results of the mutationspecific primer assay with direct sequencing was higher than that of our assay, the results were still 11.7% partially discordant and 5.0% completely discordant. The reasons why only partial genotypes could be detected using the mutation-specific primer assay might be due to: the different amplification efficiency, the difference of the reaction system between mutation-specific primer assay and direct sequencing, or its overall lower sensitivity. It is a frequent phenomenon that viruses with mixed and mutated status often exist in patients with chronic hepatitis B, and it is true that the small percentage of minorly mutated viruses are not detected by many assays. Therefore, it is important to establish a sensitive method to detect mutations as early as possible while the mutant viruses are still a minor fraction of the total HBV population. Although our method had a similar detection limit (10% for a minor population) to that of the aforementioned study by Hua et al. [26], the detection limit of our method could still reach 10% even when the virus load was between 103 and 108 copies/ml. Our method thus enables the early detection of drug-
Z. Feng et al. / Clinica Chimica Acta 412 (2011) 1018–1021
resistant mutants, implying its superiority to other methods with regard to sensitivity. In conclusion, the real-time PCR assay based on AllGlo™ probes proved to be a novel, rapid, simple, and sensitive assay to detect lamivudine-related HBV mutants even when the mutants were still a minor population. This method will be useful for the early detection of resistant mutants during lamivudine therapy.
Acknowledgments We thank Shenyou Biotechnology, Shanghai, China, for supplying the partial reagents. We are grateful to Zheng Hua-yang and Shen Jingping for technical assistance.
References [1] Zuckerman JN, Zuckerman AJ. Current topics in hepatitis B. J Infect 2000;41:130–6. [2] Bielawski KP, Stalke P. Molecular epidemiology of chronic hepatitis B virus infection in northern Poland. J Clin Virol 2005;34(Supple 1):S63–9. [3] Dienstag JL, Schiff ER, Wright TL, et al. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999;341:1256–63. [4] Lok AS, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology 2003;125:1714–22. [5] Liaw YF, Sung JJ, Chow WC, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004;351:1521–31. [6] Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B: Asia Hepatitis Lamivudine Study Group. N Engl J Med 1998;339:61–8. [7] Schalm SW, Heathcote J, Cianciara J, et al. Lamivudine and alpha interferon combination treatment of patients with chronic hepatitis B infection: a randomised trial. Gut 2000;46:562–8. [8] Liaw YF, Leung NW, Chang TT, et al. Effects of extended lamivudine therapy in Asian patients with chronic hepatitis B. Gastroenterology 2000;119:172–80. [9] Leung NW, Lai CL, Chang TT, et al. Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy. Hepatology 2001;33:1527–32. [10] Guan R, Lai CL, Liaw YF. Efficacy and safety of 5-years lamivudine treatment of Chinese patients with chronic hepatitis B. J Gastroenterol Hepatol 2001;16(Suppl 1): A60. [11] Allen MI, Deslauriers M, Andrews CW, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Hepatology 1998;27: 1670–7. [12] Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996;24:714–7.
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Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Rapid detection of the hepatitis B virus YMDD mutant using AllGlo™ probes Feng Zhao-lei a, Yu Xiao-yan b, Lu Zhi-ming c, Geng Da-ying a, Zhang Li a, Chen Shi-jun a,⁎ a b c
Department of Center Laboratory, Jinan Infectious Disease Hospital, Shandong University, Jinan, Shandong 250021, PR China Department of Laboratory Medicine, Jinan central Hospital, Jinan, Shandong 250014, PR China Department of Laboratory Medicine, Shandong Provincial Hospital, Shandong University, Jinan, Shandong 250021, PR China
a r t i c l e
i n f o
Article history: Received 31 August 2010 Received in revised form 27 January 2011 Accepted 8 February 2011 Available online 12 February 2011 Keywords: Hepatitis B virus YMDD mutant Real-time PCR AllGlo™ probe
a b s t r a c t Background: The early detection of hepatitis B virus (HBV) mutants in clinical samples is important when monitoring chronic HBV patients with lamivudine-resistant mutations during lamivudine therapy. Methods: The AllGlo™ probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The sensitivity and specificity of the assay were evaluated using a series of diluted mixtures of wild-type and mutant plasmids. This assay was compared with direct sequencing and the mutation-specific primer assay. Results: Each YMDD, YVDD, and YIDD probe only detected its corresponding plasmid. Moreover, the assay correctly identified negative samples from 40 non-HBV infected patients and 100 healthy controls. The detection limit of this assay was 50 copies/ml for YVDD and YIDD. The assay could detect the mutant strains when they were present at ≥ 10% within a mixed virus population. The assay was fully concordant with direct sequencing in 34 samples (56.7%) and partially concordant in 26 samples (43.3%), and detected more types of the HBV motif than direct sequencing. Conclusions: AllGlo™ probe assay is a novel, sensitive and specific assay to detect lamivudine-related HBV mutants, therefore, may be useful for monitoring chronic HBV patients treated with lamivudine. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a major health problem worldwide with considerable morbidity and mortality. Currently it is estimated that N2 billion people have been infected with HBV, and 300 million people have chronic HBV infection [1]. HBV is one of the leading causes of chronic liver disease, including liver cirrhosis, hepatic failure and hepatocellular carcinoma (HCC), especially in developing countries [2]. Nucleoside analogue therapy allows safe, long-term suppression of HBV and is a major milestone in the treatment of chronic hepatitis B. Lamivudine, the first-line antiviral agent approved by FDA, can effectively suppress HBV replication, reduce disease activity, improve liver histology, and delay clinical progression [3–5]. However, long-term monotherapy is usually associated with the emergence of resistant mutants, followed by increases in viral load and reelevation of aminotransferase activities. Genotypic resistance could be detected in 14% to 32% of patients after 1 year of treatment [3,6,7]. In Asia, genotypic resistance increased from 14% in year 1 to 38%, 49%, 66%, and 69% after 2–5 years of treatment, respectively [8–10]. Most lamivudine-resistant
⁎ Corresponding author at: Department of Center Laboratory, Jinan Infectious Disease Hospital, Shandong University, 173 Jingshi Rd, Jinan, Shandong 250021, PR China. Tel.: + 86 531 87924945; fax: + 86 531 87069102. E-mail address: [email protected] (S. Chen). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.012
strains are associated with mutations of the YMDD motif in the C domain of HBV polymerase. The key mutations are the substitutions of methionine at the rtM204 with either isoleucine (rtM204I, YIDD variant) or valine (rtM204V, YVDD variant) [11,12]. Several assays including fragment length polymorphism (RFLP) [13], the Line Probe Assay (INNOLiPA, Innogenetics Inc, Belgium) [14], peptide nucleic acid clamping [15], mass spectrometric analysis [16], fluorescence polarization [17], microarray [18,19], reverse dot blot [20], denature gradient gel electrophoresis [21], sequencing [22], fluorescence quantitative PCR [23], real-time PCR using mutation-specific primers [24], TaqMan probes [25], or TaqMan-minor groove binder probes [26] have been used to rapidly detect and quantify lamivudine-resistant mutations, but the sensitivity and accuracy of these methods should be further improved. In this study, we established a novel rapid, sensitive, and accurate assay based on real-time PCR using AllGlo™ probes [27] for detecting the YMDD mutations within HBV. The AllGlo™ probe has 2 identical reporter dyes that quench each other and become de-quenched once the probe is cleaved. Upon hybridization with the target sequence, the labeled oligo becomes stretched and cleaved. This leads to the separation of the two reporter dyes and consequently de-quenching occurs. Because this process releases two fluorescing molecules instead of one, the signal change is much greater, making AllGlobased quantitative PCR detections highly sensitive and robust. Using AllGlo™ probes allowed us to rapidly detect HBV mutants in clinical
Z. Feng et al. / Clinica Chimica Acta 412 (2011) 1018–1021
samples with high sensitivity and accuracy. This method may be useful for monitoring lamivudine-resistant mutations during lamivudine therapy. 2. Materials and methods 2.1. Patients and HBV DNA extraction Serum samples were collected from 60 chronic hepatitis B patients who had received lamivudine monotherapy for at least 6 months and were later found to have increased viral titers. All specimens were stored at −70 °C until tested. The levels of HBV DNA (5.67± 1.31 log copies/ ml) were measured by real-time PCR (PG Biotech, China) in an ABI-7500 thermal cycler (Applied Biosystems, Foster City, CA). Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of our hospital. HBV DNA was extracted (200 μl) in serum samples using the QIAamp blood kit (Qiagen, Hilden, Germany) and following the manufacturer's protocol. 2.2. External standards To validate the specificity and sensitivity of the assay, three external standards were constructed for the three type-specific reactions (wildtype: 204M [ATG], mutant: 204V [GTG], and mutant: 204I [ATT]). Briefly, sera with wild-type HBV DNA, YVDD, or YIDD mutants were amplified by PCR with primer sets (Table 1). The obtained PCR products were then cloned into pMD19-T Simple Vector (TakaRa, Dalian, China) using the standard protocol. Each plasmid was verified through sequencing and linearized by Sac I digestion. The copy numbers were then assessed by absorption spectrophotometry and adjusted to 1 × 101, 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 copies/ml. 2.3. Real-time PCR using AllGlo™ probes The primers and probes (Chaoshi BioTechnologies, Shanghai, China) used in this study are listed in Table 2 [26]. Three separate reaction mixtures were performed to detect wild-type YMDD, YIDD mutant, or YVDD mutant, respectively. PCR amplification was performed in a 96well plate in a 25 μl volume containing 5 μl of the extracted HBV DNA sample (or external standard), 0.4 μmol/l of a forward primer (Table 2), 0.4 μmol/l of a reverse primer, 0.2 μmol/l of a type-specific probe (YMDD and YVDD, or YIDD), and 12.5 μl of Universal PCR Master Mix (Applied Biosystems). Real-time PCR was performed on a 7500 Real-time PCR system (Applied Biosystems) and analyzed using 7500 system SDS software. All of the reactions were performed three times. The programs used were as follows: 50 °C for 1 min and 95 °C for 1 min for the initial period, and then 95 °C for 15 s followed by 62 °C for 1 min for 40 cycles. 2.4. Real-time PCR using mutation-specific primers and TaqMan probe Real-time PCR using mutation-specific primers was performed as described previously [24]. In brief, parallel reactions C, V, and I corresponding to the individual's reverse primer (Table 3) were used to detect total HBV YMDD, YVDD, and YIDD respectively.
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Table 2 Primers and probes for detection of wild-type YMDD, YVDD and YIDD mutants. Primers or probes
Sequence
Y(M/I/V)DD-F2 Y(M/I/V)DD-R2 YMDD-probe YIDD-probe YVDD-probe
5′-GGGCTTTCCCCCACTGTT-3′ 5′-GTACAGACTTGGCCCCCAATAC-3′ 5′-AllGlo-CTTTCAGTTATATGGATGATG-AllGlo-3′ 5′-AllGlo-CTTTCAGTTATATTGATGATGTG-AllGlo-3′ 5′-AllGlo-CTTTCAGTTATGTGGATGAT-AllGlo-3′
PCR was performed using 5′-TGGGCCTCAGTCCGTTTCTC-3′(HBV nt647– 666) and 5′-GGACTCAAGATGTTGTACAG-3′ (HBV nt786–767) as the sense and antisense primers, respectively, and sequenced by ABI 3100 Analyser (Applied Biosystems). 2.6. Subcloning of the HBV gene Of the samples whose detected results were partially concordant between direct sequencing and real-time PCR using AllGlo™ probes, 10 samples were selected at random for PCR amplification. The PCR products were then cloned into the pMD19-T vector and sequenced, as described by Kobayashi et al. [29]. 3. Results 3.1. Specificity of the assay Cross-reactivity tests were performed to study the specificity of the assay based on real-time PCR using AllGlo™ probes. A diluted series of each plasmid (ranging from 10 × 108 to 10× 101 copies/ml) was used as the template for PCR using AllGlo™ probes. A negative control was included to avoid nonspecific amplification. Each single YMDD, YVDD, and YIDD probe was able to only detect its corresponding plasmid. Plasmid mixtures containing the wild-type (YMDD) and mutants (YVDD and/or YIDD) between 1 × 102 and 1 × 108 copies/ml could be detected only by their corresponding probes. Also, when the assay was used to detect serum samples from 40 non-HBV infected patients and 100 healthy controls, negative results were obtained, implying the specificity and accuracy of this assay (data not shown). 3.2. Sensitivity and detection limits of the assay Mixing experiments were performed to determine the sensitivity of each probe in the assay. The YMDD plasmids and mutant plasmids (YIDD or YVDD) were mixed at ratios of 100:0, 90:10, 80:20, 50:50, 20:80, 10:90, and 0:100, corresponding to a final concentration of 108, 107, 106, 105, 104, 103, 102, and 10 copies/ml, respectively. Then the three plasmids (YMDD, YIDD, and YVDD) were mixed at ratios of 90:5:5, 80:10:10, 50:25:25, 20:40:40, 10:45:45, and 0:50:50, corresponding to the same concentration of 108, 107, 106, 105, 104, 103, 102, and 10 copies/ ml, respectively. Among the 500 copies/ml present in the mixtures, 10% of the mutants – either YVDD or YIDD – were detected. This finding demonstrates that the detection limit of the assay was 50 copies/ml.
2.5. Direct sequencing Direct sequencing of the DNA sequence of domain C of the HBV polymerase gene was carried out as described previously [28]. Briefly, Table 1 Primers for wild-type HBV DNA, YVDD and YIDD mutation amplification. Primers
Sequences
Y(M/I/V)DD-F1 Y(M/I/V)DD-R1
5′-GATGTGTCTGCGGCGTTTTA-3′ 5′-CAGCAAAGCCCAAAAGACCCAC-3′
Table 3 Probe and mutation-specific primers for YMDD, YIDD and YVDD. Denomination
Sequences
Probe Forward primer Reverse primer C Reverse primer V Reverse primer I
5′-FAM-AGCCCTACGAACCACTGAAC-TAMRA-3′ 5′-CCTATGGGAGTGGGCCTC-3′ 5′-GCCCCCAATACCACATCATC-3′ 5′-CCCAATACCACATCATCCAC-3′ 5′-CCCCCAATACCACATCATCA-3′ 5′-CCCCCAATACCACATCATCG-3′ 5′-CCCCCAATACCACATCATCT-3′
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3.3. Comparison of direct sequencing with real-time PCR using AllGlo™ probes or mutation-specific primers Three methods were compared: direct sequencing, real-time PCR using AllGlo™ probes (AllGlo™ probe assay), and real-time PCR using mutation-specific primers (mutation-specific primer assay). The results of these three methods performed on 60 serum samples from chronic hepatitis B patients receiving lamivudine monotherapy were analyzed as described by Evelien Libbrecht [30]. Each assay result was divided into four classes: fully concordant, partially concordant, partially discordant, and completely discordant with direct sequencing. Results were considered fully concordant if both direct sequencing and the AllGlo™ probe assay or the mutation-specific primer assay showed the same results. The results were considered partially concordant if the AllGlo™ probe assay or the mutation-specific primer assay provided additional information compared to that provided by direct sequencing. For example, the AllGlo™ probe assay and the mutation-specific primer assay showed a mixture of wild-type and mutant sequences, while direct sequencing showed only a wild-type or a mutant sequence. The results were considered partially discordant if direct sequencing showed a mixture of wild-type and mutant sequences, while the AllGlo™ probe assay and the mutation-specific primer assay showed a wild-type sequence or a mutant sequence only. The results were considered completely discordant if one test showed a mutant and the other test resulted in a wild type. As demonstrated in Table 4, the results produced by the AllGlo™ probe assay and direct sequencing were fully concordant in 34 samples (56.7%) whether it was a single-type or a mixed-type. The results were partially concordant in 26 samples (43.3%). In all partially concordant samples, the AllGlo™ probe assay detected more types of motifs (YMDD, YVDD, or YIDD) than that provided by direct sequencing, meaning that our assay provided more information. Of these 26 samples, 10 samples were selected at random to amplify and subclone. Then 20 randomly selected clones per sample were picked and sequenced. The results produced by subcloning were fully concordant with that produced by our AllGlo™ probe assay. The results produced by the mutation-specific primer assay and direct sequencing were fully concordant in 47 samples (78.3%), partially concordant in 3 samples (5.0%), partially discordant in 7 samples (11.7%), and completely discordant in 3 samples (5.0%), respectively. The results produced by the three methods were fully concordant in 27 samples (45.0%) (Table 4). 4. Discussion While new and effective nucleotide/nucleoside analogues are increasingly available for the treatment of chronic HBV infection, for reasons of efficacy, safety, and cost, lamivudine remains the first-line therapy in developing countries with a high incidence of chronic HBV infection. However, the beneficial effects of antiviral agents in halting disease progression are blunted by the occurrence of drug mutations. Severe hepatitis reactivation due to drug-resistant virus can result in hepatic decompensation and even mortality. Such unsatisfactory out-
Table 4 Concordant results obtained through blind analysis using three methods of 60 serum samples. Type
Sequencing vs. AllGlo™ probe assay
Sequencing vs. mutation-specific primer assay
Fully concordant Partially concordant Partially discordant Completely discordant Total
34 26
47 3 7 3
60
60
comes could be prevented through early detection, thereby allowing clinicians to select appropriate alternatives. In this study, we described a real time PCR based on AllGlo™ probes for genotyping the YMDD motif variants in clinical samples from HBVinfected individuals. This new approach has several potential advantages over other methods. First, this assay is more sensitive than direct sequencing or a mutation-specific primer assay. The detection limit of the real time PCR based on AllGlo™ probes is 50 copies/ml for YVDD and YIDD with 10% sensitivity within a mixed virus population. This assay also has high specificity. It could detect YMDD mutants at a copy number ranging from 50 to 108. Due to its wide detection range, this assay can be applied in a clinical context for detection of YMDD mutants having a low amplification efficiency. Hua et al. [26] reported a novel, rapid, sensitive, and accurate method for detecting YMDD mutants using TaqMan-minor groove binder probes (MGB) probes, which have a fluorophore at the 5′-end and a nonfluorescent quencher at the 3′-end. These probes can produce very low background and strong hybridization-triggered fluorescence. This method's detection limit is 10 copies for YVDD and 50 copies/ml for YIDD with 10% sensitivity within a mixed virus population. Using the AllGlo™ probes, the detection limit of our assay is 50 copies/ml for both YVDD and YIDD. The AllGlo™-based quantitative PCR detections are highly sensitive and theoretically robust because the AllGlo™ probe has two identical reporter dyes that quench each other and become dequenched once the probe is cleaved. When de-quenching occurs, two fluorescing molecules are released, instead of one. This creates a much greater signal change. Therefore, the detection limit of our assay is theoretically more sensitive than that of the method using TaqManMGB probes. We inferred that the lower sensitivity of our assay might be associated with the quality and/or quantity of nucleotides extracted from plasmids. To compare the concordance of direct sequencing with the AllGlo™ assay or the mutation-specific primer assay, 60 clinical samples were analyzed using three methods: direct sequencing, AllGlo™ probe assay, and mutation-specific primer assay. Fully concordant results of the AllGlo™ probe assay with direct sequencing were observed in 34 samples (56.7%). Partially concordant results were observed in 26 samples. Neither partially discordant nor completely discordant results were observed. Moreover, there were more genotypes detected by this assay than by direct sequencing in the partially concordant samples. Results from subcloning experiments conducted on 10 randomly selected samples were consistent with this assay, suggesting the accuracy and superiority of our novel method. Although direct sequencing had previously been considered the “gold standard” due to its accuracy, its sensitivity is limited (approximately 20% of the total virus population) [26]. The ability to detect only partially mixed genotypes might be associated with its relatively low sensitivity. The mutation-specific primer assay is also a sensitive method for detecting YMDD mutants. Although the fully concordant results of the mutationspecific primer assay with direct sequencing was higher than that of our assay, the results were still 11.7% partially discordant and 5.0% completely discordant. The reasons why only partial genotypes could be detected using the mutation-specific primer assay might be due to: the different amplification efficiency, the difference of the reaction system between mutation-specific primer assay and direct sequencing, or its overall lower sensitivity. It is a frequent phenomenon that viruses with mixed and mutated status often exist in patients with chronic hepatitis B, and it is true that the small percentage of minorly mutated viruses are not detected by many assays. Therefore, it is important to establish a sensitive method to detect mutations as early as possible while the mutant viruses are still a minor fraction of the total HBV population. Although our method had a similar detection limit (10% for a minor population) to that of the aforementioned study by Hua et al. [26], the detection limit of our method could still reach 10% even when the virus load was between 103 and 108 copies/ml. Our method thus enables the early detection of drug-
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resistant mutants, implying its superiority to other methods with regard to sensitivity. In conclusion, the real-time PCR assay based on AllGlo™ probes proved to be a novel, rapid, simple, and sensitive assay to detect lamivudine-related HBV mutants even when the mutants were still a minor population. This method will be useful for the early detection of resistant mutants during lamivudine therapy.
Acknowledgments We thank Shenyou Biotechnology, Shanghai, China, for supplying the partial reagents. We are grateful to Zheng Hua-yang and Shen Jingping for technical assistance.
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