LETTER
Abe et al. reported the development of a real-time PCR assay for the absolute quantification of the hepatitis B virus (HBV) genome by TaqMan probe-based chemistry in 1999 (1). Their studies included two set of primers and probes, and the primer named HBSR1 (5′-GGTGAGTGATTGGAGGGTTG-3′) was found to be carrying an additional guanidine nucleotide (underlined) near the 3′ end, resulting in a primer mismatch (Fig. 1).
Fig. 1.
Alignment of 16 HBV S genes. Partial length data are shown with the primer HBSR1 sequence depicted from the 5′ end to the 3′ end. A primer mismatch of HBSR1 was found, with the addition of a guanidine nucleotide close to the 3′ end. Nucleotides that diverge from the consensus are underlined.
We have duplicated the experiment with an identical setup and protocol and found that the addition of a single nucleotide in the HBSR1 primer significantly reduced the limit of detection of the assay, and more importantly, it could cause a false-negative diagnostic outcome in certain clinical screening procedures with particularly low viral copy numbers in the specimens (3).
The consequence of the primer mismatch was not significant in a higher-viral-load control (Fig. 2). However, the diagnostic assay with primer HBSR1 did not show a positive signal with an HBV viral load of 1 × 10 IU/ml (AcroMetrix, Benicia, CA) and was found to give a false-negative result compared to both the corrected primer sequence HBSR1c (5′-GGTGAGTGATTGGAGGTTG-3′) and our in-house HBV diagnostic assay (Fig. 2). The observation of this primer mismatch was also reported in another two publications (2, 4), and such mistakes should not be neglected. Therefore, we urgently call for an in-depth correction of this mistake to avoid the potential of missing HBV-positive samples or invalid diagnosis of HBV specimens in the field.
Fig. 2.
Real-time PCR detection of an HBV control serum panel (2 × 103 IU/ml and 2 × 10 IU/ml) with primers HBSR1 and HBSR1c and our in-house diagnostic assay. The amplification profile strictly followed that of Abe et al., with initial UNG and Taq activation at 50°C for 2 min, 95°C for 10 min, and 55 cycles of amplification of 95°C for 20 s and 60°C for 1 min. Fluorescence signals were acquired at 60°C during the elongation step. ΔRn, the magnitude of the PCR signal.
Footnotes
For the authors' reply, see doi:10.1128/JCM.05516-11.
Contributor Information
Chun-Kin Chow, Hai Kang Life Corporation Limited, Shau Kei Wan, Hong Kong SAR, China.
Kun Qin, Chinese National Influenza Center, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Lok-Ting Lau, Hai Kang Life Corporation Limited, 8/F Hang Tung Resources Centre, 18 A-Kung Ngam Village Road, Shau Kei Wan, Hong Kong SAR, China.
Albert Cheung-Hoi Yu, Neuroscience Research Institute, Department of Neurobiology, School of Basic Medical Sciences, Peking University, 38 Xue Yuan Road, Beijing 100191, China.
REFERENCES
- 1. Abe A., et al. 1999. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J. Clin. Microbiol. 37:2899–2903 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Ghisetti V., et al. 2004. Occult hepatitis B virus infection in HBsAg negative patients undergoing liver transplantation: clinical significance. Liver Transpl. 10:356–362 [DOI] [PubMed] [Google Scholar]
- 3. Kleinman S. H., et al. 2003. Frequency of HBV DNA detection in US blood donors testing positive for the presence of anti-HBc: implications for transfusion transmission and donor screening. Transfusion 43:696–704 [DOI] [PubMed] [Google Scholar]
- 4. Li K. S., et al. 2001. Hepatitis B virus harboring nucleotide deletions in the core promoter region and genotype B correlate with low viral replication activity in anti-HBe positive carriers. J. Clin. Virol. 23:97–106 [DOI] [PubMed] [Google Scholar]