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Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal.

ABSTRACT: We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR. Six additional cell culture-negative samples were positive by PCR with both sets of primers. Total processing time was less than 3 h. Real-time PCR using SYBR Green I as detection signal is a sensitive procedure for the rapid diagnosis of HSV in genital lesions.

SUBMITTER: Aldea C

PROVIDER: S-EPMC120289 | biostudies-literature | 2002 Mar

REPOSITORIES: biostudies-literature

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Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal.

Aldea Carmen C Alvarez Carmen P CP Folgueira Lola L Delgado Rafael R Otero Joaquín R JR

Journal of clinical microbiology 20020301 3

We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR ...[more]

PMID: 11880439

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Project description:BACKGROUND: The speed and sensitivity of real-time polymerase chain reaction (PCR) have made it a popular method for the detection of microbiological agents in both research and clinical specimens. For the detection and genotyping of herpes simplex virus (HSV) in clinical specimens, real-time PCR has proven to be faster, more sensitive and safer than earlier methods which included isolation of the virus in cell culture followed by immunofluorescence microscopy. While PCR-based assays for HSV detection posses clear advantages over these earlier techniques, certain aspects of the PCR method remain onerous. The process of extraction and purification of nucleic acid from clinical specimens prior to PCR is particularly cumbersome. Nucleic acid extraction is expensive, time-consuming and provides a step whereby specimens can become contaminated prior to their analysis. Herein, we investigate the necessity of nucleic acid extraction from swab-based clinical specimens for HSV detection by real-time PCR. We find that nucleic acid extraction is unnecessary for specific and sensitive detection of HSV in clinical specimens using real-time PCR. METHODS: Prospective (n = 36) and retrospective (n = 21) clinical specimens from various anatomical sites were analyzed for the presence of herpes simplex virus 1 or 2 by real-time PCR using the RealArt HSV 1/2 LC PCR Kit. Specimens were analyzed by PCR both before and following automated nucleic acid extraction. PCR using extracted and unextracted specimens was also compared to cell culture as a means of detecting HSV. RESULTS: Detection of HSV 1/2 DNA in clinical specimens by real-time PCR did not require that the specimen be subjected to nucleic acid extraction/purification prior to analysis. Each specimen that was detectable by real-time PCR when analyzed in the extracted form was also detectable when analyzed in the unextracted form using the methods herein. The limit of detection of HSV-1 and HSV-2 particles when analyzed in the unextracted form was found to be approximately 17 and 32 virus particles respectively, compared to a sensitivity of 10 copies, for analysis of purified DNA. Omission of the nucleic acid extraction step shortened both the assay time and cost. CONCLUSION: Omission of the nucleic acid extraction step prior to real-time PCR for detection of herpes simplex virus resulted in a more rapid and cost-effective assay, with little impact upon the sensitivity of detection.

Project description:This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 102-1.31 × 107 copies/?L. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R2) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method for ducks infected with duck circovirus.

Dataset Information

Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal.

Publications

Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal.

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