PubMed · 11243893
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M Carbone. 2001. Introduction.. https://doi.org/10.1006/scbi.2000.0340
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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.
Liquid based cytology samples are being used increasingly to improve cervical screening and have the advantage that residual cell suspension is available for other tests such as human papillomavirus (HPV) detection. However, as the transport medium is optimised for downstream cytology, problems can be experienced during extraction of nucleic acid. This study aimed to develop a robust protocol for automated extraction of HPV DNA from cervical, liquid based cytology samples using a high throughput robotic system. Considerable modification of existing clinical extraction protocols for swab specimens, together with optimisation of required sample input volume was required to reduce sample blockage during the extraction to acceptable levels. The blockage rate and optimal processing volume was assessed by extracting a fixed volume (1/4) of re-suspended material from the centrifuged pellets of 10, 5 and 1 ml aliquots of 200 specimens. Analysis revealed 17.5% blockage with specimens originating from 10 ml aliquots; 3% with 5 ml and no blockage with 1 ml aliquots of the same samples. A 3% blockage level is acceptable for an automatic well clearance procedure to be followed. HPV testing of the extracts by real-time PCR showed a 1.5% loss of sensitivity in extracts originating from 1 ml aliquots as compared with 5 ml aliquots with a consequent loss of detectable HPV genotypes after reverse hybridisation. In short, 5 ml of liquid based cytology specimen is recommended for nucleic acid extraction, to allow optimal detection of HPV types in clinical samples while retaining maximum efficiency of the robotic extraction procedure.
The measurement of the Epstein-Barr viral load in peripheral blood has been recognised as an important way of monitoring the response to treatment in patients with Epstein-Barr virus (EBV)-related malignancies. In particular, EBV load in transplant recipients can be used as a predictive parameter for Post-transplant Lymphoproliferative Disorder (PTLD). The aim was to develop a rapid and reliable PCR protocol for the quantification of the cell-associated EBV genome. Real-time PCR using TaqMan methodology was established. This technique was applied to determine the EBV load in various study groups including healthy controls, transplant recipients, patients on haemodialysis, and patients with infectious mononucleosis. The baseline level of EBV genomes in the immunosuppressed renal transplant recipients was significantly different from that in the healthy controls.