| Rapid Extraction of DNA using Ion Exchange Resin for Early Detection ofMycobacterium tuberculosis by the Polymerase Chain Reaction. |
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Cheol Min Kim, Seung Kyu Park, Mal Hyun Shon, Young Kim, Sun Dae Song, Eun Sook Jun, Han Chul Son, Byung Sun Jung |
1Institute of Clinical Research, National Masan Tuberculosis Hospital, Masan,, Korea.
2Institute of Pathology, Medical Institute, Pusan National University Hospital, Pusan, Korea.
3Department of Clinical Pathology, Department of Biochemistry, Medical College, Pusan National University, Pusan, Korea. |
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| Abstract |
BACKGROUND
The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(Polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. METHODS: We used the InstaGene(TM) DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1, 245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2, 536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. RESULTS: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value.. CONCLUSION: Even though both methods had lower possibility of cross contamination, shorter time requrirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usfulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples. |
| Key Words:
Tuberculosis, PCR, InstaGene, Proteinase K, Microwave |
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