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Recovery efficiences on nucleic acid extraction kits as measured by quantitative LightCycler PCR.

ABSTRACT: AIMS:To compare the efficiency of five nucleic acid extraction kits for processing clinical material for the diagnosis of infection. METHODS:Five nucleic acid extraction kits for processing clinical material for the diagnosis of infection were compared for their relative efficiencies in purifying and recovering either viral DNA or RNA from serum samples. Quantitative polymerase chain reaction (PCR) assays for hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were performed on the Light-Cycler instrument to determine the relative concentrations of the viral nucleic acids recovered by the various protocols. RESULTS:Large differences between kits in recovery efficiencies were observed for HBV DNA, with those protocols using enzyme digestion in addition to chaotropic solutions performing better than those using chemical disruption alone. CONCLUSIONS:There were large differences between the kits and it appeared that those extraction kits containing a proteolytic enzyme and carrier nucleic acid (and that are supplied RNase free) have the widest potential application in the routine microbiology laboratory.

SUBMITTER: Read SJ

PROVIDER: S-EPMC1187008 | biostudies-literature | 2001 Apr

REPOSITORIES: biostudies-literature

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Recovery efficiences on nucleic acid extraction kits as measured by quantitative LightCycler PCR.

Read S J SJ

Molecular pathology : MP 20010401 2

<h4>Aims</h4>To compare the efficiency of five nucleic acid extraction kits for processing clinical material for the diagnosis of infection.<h4>Methods</h4>Five nucleic acid extraction kits for processing clinical material for the diagnosis of infection were compared for their relative efficiencies in purifying and recovering either viral DNA or RNA from serum samples. Quantitative polymerase chain reaction (PCR) assays for hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were perform ...[more]

PMID: 11322169

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Project description:BackgroundThe 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.MethodsProspective (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.ResultsDetection 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.ConclusionOmission 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.

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Recovery efficiences on nucleic acid extraction kits as measured by quantitative LightCycler PCR.

Publications

Recovery efficiences on nucleic acid extraction kits as measured by quantitative LightCycler PCR.

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