Improved Blood Viral Detection with CleanAmp® Primers
September 2014
The development of reliable, high sensitivity assays for viral infection is of the utmost importance for minimizing the prevalence of infections related to the transfusion of infected blood. While original viral detection assays detect antibodies to the virus, these assays are being replaced by nucleic acid tests (NATs), which detect viral RNA or Viral DNA. NATs have been shown to display improved sensitivity as compared to antibody-based assays, allowing for earlier detection of viral infection in a blood donor. In Asia, Hepatitis B virus (HBV) is prevalent, with greater than 10% of the population testing positive for the core HBV antibody. This high infection rate is further complicated by the presence of occult HBV infection, which accounts for a 0.9% rate of acute HBV infection arising from blood transfusions. This rate of infection is up to 40 times higher than in other countries, obligating the need for a more sensitive assay for HBV infection.
To address this need, a team of researchers from South China University, Taitaigen, Inc. and the Schenzhen Blood Bank has worked together to develop a triplex NAT for simultaneous detection of HBV, Hepatitis C virus (HCV), and Human Immunodeficiency Virus, type 1 (HIV-1) in human plasma samples. This work was led by Dr. Xinglong Xiao, a scientist with ten years of experience in diagnostic assay development, and is published in the Journal of Virological Methods. The developed assay is a one-step reverse transcription-PCR (RT-PCR) assay which employed TriLink CleanAmp Primers to block the extension of PCR primers which were not essential to the reverse transcription step. This resulted in development of a sensitive and specific in-house triplex NAT, which was able to detect a panel of seven HBV genotypes at levels as low as 10 IU/mL. This assay was applied to a panel of 33,025 Human plasma samples, which tested negative in an antibody-based assay for the three viruses. Of these samples, 53 tested positive in either the triplex NAT or a commercially available NAT, with 51 samples (0.15%) testing positive for HBV. From the 51 positive HBV samples, 11 (21%) were detected by both assays, 12 (23%) were detected by the commercial assay alone, and 28 (55%) were detected by the in-house NAT alone. These findings indicate the importance of the development of a sensitive NAT assay for HBV which will detect all prevalent viral genotypes with high sensitivity. Furthermore the use of CleanAmp Primers in this study provided better control of the reverse transcription step, allowing for greater specificity and sensitivity of one-step RT-PCR.
The CleanAmp Primers used in these studies contain thermolabile phosphotriester modification groups, which were used to improve the sensitivity and specificity of PCR amplification. CleanAmp Primer technology offers a unique primer-based Hot Start activation approach, and provides benefits in routine PCR and in one-step RT-PCR. CleanAmp Primers are available through TriLink, and the precursor phosphoramidite is available from Glen Research.
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