PubMed · 4873813
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B Woodson. 1968. Recent progress in poxvirus research.. https://doi.org/10.1128/br.32.2.127-137.1968
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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.
HBV DNA quantitation is used extensively for the monitoring of treatment of hepatitis B virus (HBV) infection. The aim of this study was to develop a highly sensitive and reproducible real-time PCR (RTD-PCR) assay for the quantitation of HBV DNA using the LightCycler system. The performance of this assay was assessed by analyzing serial dilutions of HBV genomic DNA of known concentration and the lower limit of detection was found to be 1 DNA copy/reaction. By using serial dilutions of plasmid standard, RTD-PCR was determined to quantify HBV DNA in a 10-log10 dynamic range. RTD-PCR was found to be more sensitive than the commercially available tests such as the Quantiplex HBV DNA and the AMPLICOR HBV MONITOR assays. The median coefficient of variation of interexperimental variability was 3.2%. The HBV DNA values obtained with RTD-PCR were highly correlated with assays available commercially. These findings suggest that our RTD-PCR assay combines high sensitivity and reproducibility for HBV DNA quantitation in an incomparable high dynamic range of quantitation.
A conjugate of a triple helix forming oligonucleotide (TFO) and the Lambda and Delta enantiomers of the ruthenium diphenanthroline dipyridophenazine complex [Ru(phen)(2)dppz](2+) was synthesized. The ruthenium complex was attached to the 5'-end of the TFO through the dppz moiety. This conjugate formed a stable triple helix with the polypurine tract (PPT) sequence from HIV proviral DNA. The thermal denaturation temperature of the triplex was increased by 12 degrees C. One remarkable property of the Delta-[Ru(phen)(2)dppz](2+) complex is a strong increase in its fluorescence when it intercalates into DNA. While the fluorescence of the oligonucleotide conjugate was very weak, the formation of a duplex with a complementary sequence or of a triple helix with a target duplex resulted in a large increase in fluorescence of the Delta enantiomer. The increase in fluorescence allowed us to follow the kinetics of duplex and triplex formation by fluorescence spectrometry. In contrast, the Lambda enantiomer gave a much smaller fluorescence change when a triplex was formed, even though the stability of the triplex was comparable to that of the Delta enantiomer. The property was ascribed to intercalation of the dipyridophenazine moiety of the Delta enantiomer into DNA and subsequent threading of the ruthenium complex through the DNA double helix. Salt effects were consistent with the involvement of DNA breathing in the formation of the intercalating complex.