A T Kalghatgi1, A K Praharaj2, A K Sahni3, D Pradhan4, S Kumaravelu5, P L Prasad6, A Nagendra7. 1. Senior Advisor (Pathology), Command Hospital (Central Command), Lucknow. 2. PDMS (HS) DGMS Navy, Naval HQ, New Delhi. 3. Senior Advisor (Pathology), AH(R&R), Delhi Cantt. 4. Post Graduate Trainee (Department of Microbiology), AFMC Pune-40. 5. Comdt, 155 BH, C/o 99 APO. 6. Senior Advisor (Paediatrics), Command Hospital (Central Command), Lucknow. 7. Ex- Dy Comdt, Command Hospital (Western Command), Chandigarh.
Abstract
BACKGROUND: Polymerase chain reaction (PCR) is useful for rapid microbial detection in body fluids with low microbial load. It is easier to use universal or broad range primers for the amplification of conserved stretches of DNA common to all bacteria like 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) of PCR products. METHODS: Forty samples of cerebrospinal fluid were collected. After DNA extraction, universal or broad range PCR was performed using two universal primers U1-5'-CCAGCAGCCGCGGTAATACG-3', corresponding to nucleotides 518 to 537 of the Escherichia coli 16S rRNA gene, and U2 - 5'-ATCGG(C/T)TACCTTGTTACGACTTC-3', corresponding to nucleotides 1513 to 1491 of the same gene. The PCR product was subjected to digestion by endonucleases- HaeIII, Mn11, BstB1 and Alu1. Restriction pattern obtained was compared with that of standard organisms to identify the pathogen. The results were compared with conventional methods. RESULT: Universal PCR could detect pathogens in 20% samples within 13-18 hours as compared to 16% by conventional methods. The analytical sensitivity was 10 Gram negative and 250 Gram positive organisms per 200 μl sample. Overall sensitivity was 83.3% and specificity was 91.2%. CONCLUSION: Universal PCR followed by RFLP of PCR product is a good alternative to conventional diagnosis of bacterial pathogens.
BACKGROUND: Polymerase chain reaction (PCR) is useful for rapid microbial detection in body fluids with low microbial load. It is easier to use universal or broad range primers for the amplification of conserved stretches of DNA common to all bacteria like 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) of PCR products. METHODS: Forty samples of cerebrospinal fluid were collected. After DNA extraction, universal or broad range PCR was performed using two universal primers U1-5'-CCAGCAGCCGCGGTAATACG-3', corresponding to nucleotides 518 to 537 of the Escherichia coli 16S rRNA gene, and U2 - 5'-ATCGG(C/T)TACCTTGTTACGACTTC-3', corresponding to nucleotides 1513 to 1491 of the same gene. The PCR product was subjected to digestion by endonucleases- HaeIII, Mn11, BstB1 and Alu1. Restriction pattern obtained was compared with that of standard organisms to identify the pathogen. The results were compared with conventional methods. RESULT: Universal PCR could detect pathogens in 20% samples within 13-18 hours as compared to 16% by conventional methods. The analytical sensitivity was 10 Gram negative and 250 Gram positive organisms per 200 μl sample. Overall sensitivity was 83.3% and specificity was 91.2%. CONCLUSION: Universal PCR followed by RFLP of PCR product is a good alternative to conventional diagnosis of bacterial pathogens.
Entities:
Keywords:
Body fluids; Polymerase chain reaction; Restriction fragment length polymorphism
Authors: Georg Mitterer; Martin Huber; Ernst Leidinger; Claudia Kirisits; Werner Lubitz; Manfred W Mueller; Wolfgang M Schmidt Journal: J Clin Microbiol Date: 2004-03 Impact factor: 5.948