Simultaneous Visual Detection of Multiple Viral Amplicons by Dipstick Assay

ABSTRACT A sensitive, simple, and instrument-independent method for the visual detection and identification of multiple nucleic acid amplicons by dipstick has been developed. This method is based on nucleic acid hybridization on the dipstick membrane and a signal amplification system to allow visual detection. With hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) as model analytes, it is demonstrated that the visual dipstick test combined with multiplex reverse transcription (RT)-PCR for the amplification of viral nucleic acid provides a specific and sensitive detection method. The RT-PCR products were detected by the dipstick with an efficiency similar to that of a complex, expensive, and instrument-dependent method based on fluorogenic oligonucleotide probes. The detection limits of the dipstick combined with multiplex RT-PCR were 50, 125, and 500 IU/ml for HBV DNA, HCV RNA, and HIV-1 RNA, respectively. The dipstick assay detected with similar efficiencies amplicons derived from strains of HBV genotypes A through F, HCV genotypes 1 to 6, and HIV-1 subtypes A through H as well as CRF02 circulating recombinant forms of HIV-1. Analysis of 295 clinical samples and 19 pools of 10 plasma specimens from blood donors revealed that multiplex dipstick detection was reproducible, sensitive, and specific. The visual dipstick detection of multiple amplicons thus provides an attractive alternative to complex, instrument-dependent detection methods currently in use for nucleic acid testing. This new and sensitive method for nucleic acid detection should increase the availability of genomic screening in resource-limited settings and its applicability to near-patient testing.

[1]  A. Malcolm,et al.  Nucleic acid analysis by sandwich hybridization , 1989, Journal of clinical laboratory analysis.

[2]  R. Durst,et al.  Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes. , 1996, Clinical chemistry.

[3]  M. Mondelli,et al.  Clinical significance of hepatitis C virus genotypes. , 1999, Journal of hepatology.

[4]  V. Shyamala,et al.  The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana. , 2003, Blood.

[5]  J. Temple,et al.  Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1. , 2004, Journal of virological methods.

[6]  G. Schreiber,et al.  Comparative sensitivity of HBV NATs and HBsAg assays for detection of acute HBV infection , 2003, Transfusion.

[7]  T. Hirose,et al.  Automated Multiplex Assay System for Simultaneous Detection of Hepatitis B Virus DNA, Hepatitis C Virus RNA, and Human Immunodeficiency Virus Type 1 RNA , 2001, Journal of Clinical Microbiology.

[8]  H. Niesters,et al.  Development of a real-time quantitative RT-PCR for the detection of HIV-2 RNA in plasma. , 2000, Journal of virological methods.

[9]  A. Heath,et al.  An international collaborative study to establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based techniques. , 2001, Journal of virological methods.

[10]  F. Brun-Vézinet,et al.  HIV-1/HIV-2 seronegativity in HIV-1 subtype 0 infected patients , 1994, The Lancet.

[11]  H. Ikeda,et al.  Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay , 2001, Transfusion.

[12]  Mats Nilsson,et al.  Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay. , 2003, Analytical biochemistry.

[13]  M Herzberg,et al.  A novel rapid hybridization technique: paper chromatography hybridization assay (PACHA). , 1993, Gene.

[14]  J. Nkengasong,et al.  The puzzle of HIV‐1 subtypes in Africa , 1997, AIDS.

[15]  K. Nishioka,et al.  High throughput screening of 16 million serologically negative blood donors for hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1 by nucleic acid amplification testing with specific and sensitive multiplex reagent in Japan. , 2003, Journal of virological methods.

[16]  E. Seifried,et al.  Feasibility and efficacy of routine PCR screening of blood donations for hepatitis C virus, hepatitis B virus, and HIV-1 in a blood-bank setting , 1999, The Lancet.

[17]  A. Heath,et al.  Collaborative study to calibrate hepatitis C virus genotypes 2–6 against the HCV International Standard, 96/790 (genotype 1) , 2003, Vox sanguinis.

[18]  S. Glynn,et al.  Frequency of HBV DNA detection in US blood donors testing positive for the presence of anti‐HBc: implications for transfusion transmission and donor screening , 2003, Transfusion.

[19]  A. Kidd,et al.  Genetic variability in hepatitis B viruses. , 2002, The Journal of general virology.

[20]  R. Lanciotti,et al.  Nucleic Acid Sequence-Based Amplification Assays for Rapid Detection of West Nile and St. Louis Encephalitis Viruses , 2001, Journal of Clinical Microbiology.

[21]  A. Hall,et al.  Estimation of the risk of hepatitis B virus, hepatitis C virus and human immunodeficiency virus infectious donations entering the blood supply in England, 1993–2001 , 2003, Vox sanguinis.

[22]  B. Cant,et al.  Evaluation of a transcription‐mediated amplification‐based HCV and HIV‐1 RNA duplex assay for screening individual blood donations: a comparison with a minipool testing system , 2003, Transfusion.

[23]  R A Durst,et al.  Detection of Cryptosporidium parvum using oligonucleotide-tagged liposomes in a competitive assay format. , 2001, Analytical chemistry.

[24]  J. Allain,et al.  Occult hepatitis B virus infection: implications in transfusion , 2004, Vox sanguinis.

[25]  Helen H. Lee,et al.  AIDS in an HIV‐seronegative Ghanaian woman with intersubtype A/G recombinant HIV‐1 infection , 2000, Journal of medical virology.

[26]  A. Heath,et al.  An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques , 2001, Vox sanguinis.

[27]  A. Baeumner,et al.  Biosensor for dengue virus detection: sensitive, rapid, and serotype specific. , 2002, Analytical chemistry.

[28]  V. Shyamala,et al.  Detection and quantitation of HBV DNA in the WHO International Standard for HIV-1 RNA (NIBSC code: 97/656). , 2004, Journal of virological methods.

[29]  R. Durst,et al.  Characteristics of DNA-tagged liposomes allowing their use in capillary-migration, sandwich-hybridization assays. , 1997, Analytical biochemistry.

[30]  P E Klapper,et al.  Multiplex PCR: Optimization and Application in Diagnostic Virology , 2000, Clinical Microbiology Reviews.

[31]  Helen H. Lee,et al.  Molecular epidemiology of HIV in Ghana: Dominance of CRF02_AG , 2004, Journal of medical virology.

[32]  R. Tedder,et al.  Evidence that anti‐HBc but not HBV DNA testing may prevent some HBV transmission by transfusion , 1999, British journal of haematology.

[33]  J. Temple,et al.  Frequent Recovery and Broad Genotype 2 Diversity Characterize Hepatitis C Virus Infection in Ghana, West Africa , 2003, Journal of Virology.

[34]  Maurice Rosenstraus,et al.  An Internal Control for Routine Diagnostic PCR: Design, Properties, and Effect on Clinical Performance , 1998, Journal of Clinical Microbiology.