Automated Multiplex Assay System for Simultaneous Detection of Hepatitis B Virus DNA, Hepatitis C Virus RNA, and Human Immunodeficiency Virus Type 1 RNA

Abstract

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (PerkinElmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).

Infectious Agents Transmitted by Blood and Blood Component and Impacts of Nükleik Asit Amplifikasyon Test (NAT)

“…HCV araştırmasın-da kullanılan kitlerin paraproteinemi, üremi, RF pozitifliği gibi durumlarda yalancı pozitifliği de söz konusudur (35) . HCV antijen testi ve NAT kullanımı ile kan güvenliğinin arttırılması sağlanabilir (20,(22)(23)(24)(25)(26)(27)(28)(29)(30)36) . Amerika Birleşik Devletlerinde NAT kullanımı sonrası kan ve bileşenleri ile HCV bulaş riski 1:1,935,000 ünite olarak hesaplanmaktadır (21) .…”

“…NAT sistemlerin etkinliğinin araştırıldığı çok sayıda karşı-laştırmalı çalışma mevcuttur. NAT'ın transfüzyon bulaş riskini azalttığını, ancak sıfırlayamadığını da belirtmek gerekir (20,(22)(23)(24)(25)(26)(27)(28)(29)(30) .…”

Simultaneous detection of HBV and HCV by multiplex PCR normalization

“…The levels of concordance with the data obtained by conventional single PCR method were 100% for single infection, 98-100% for double infection, and 92% for triple infection. Meng Q and his colleagues [9] established an automatic multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The detection limits (95% confidence interval) were 22 to 60 copies/mL for HBV, 61 to 112 IU/mL for HCV, and 33 to 66 copies/mL for HIV-1, using a specimen input volume of 0.2 mL.…”

Correspondências

“…A média de detecção para HIV1-RNA foi de 14 dias antes do anticorpo HIV1 e uma média de nove dias antes do antígeno p24 6 .…”