PubMed · 1822521
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W Gerlich. 1991. Hepatitis B surface proteins.. https://doi.org/10.1016/0168-8278(91)90033-8
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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.
The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression.
Neoplastic and non-neoplastic tissues from the urinary tract and the prostate were analyzed for the presence of human papillomavirus (HPV) DNA. The analysis was performed by PCR using primers specific for HPV 6/11 and 16. HPV DNA was present in bladder, ureter, kidney and prostate, with percentages ranging between 46% and 87%. Benign and oncogenic HPV types were detected with similar frequencies both in non-neoplastic and in neoplastic biopsies, and HPV 16 was not preferentially associated with malignant lesions. In all instances, small amounts of HPV DNA were present in the tissues, suggesting the absence of productive infection. Analysis of the physical state of HPV DNA performed by 2-dimensional gel electrophoresis and Southern blot hybridization revealed that HPV 16 DNA harbored in the urinary tract can be integrated also in non-neoplastic tissues. The results indicate that HPV 16 does not seem to be associated with urinary-tract and prostate oncogenesis, but that these tissues may represent an important reservoir for the transmission of HPV types normally infecting the genital tract.