Detection of DNA in blood samples using direct-PCR test | ||
| Egyptian Pharmaceutical Journal | ||
| Volume 12, Issue 2, July 2013, Pages 115-119 PDF (339.31 K) | ||
| DOI: 10.4103/1687-4315.124005 | ||
| Authors | ||
| Ahmed M. Mora; Nour M. Abdel-Kader; Soheir S. Maklad | ||
| Abstract | ||
| Background Meningococcal disease caused by is a widely distributed complex human disease affecting all age categories. Successful and effective treatment of patients with this disease depends on precise and early diagnosis of the disease. Objective The aim of this study was to evaluate the possible use of direct PCR (D-PCR) for the detection and amplification of DNA in blood samples compared with the conventional PCR (C-PCR) method, which needs bacterial culture and DNA extraction. Materials and methods Specific primers on the basis of the 16S rDNA of were used for the amplification of 600 bp DNA fragment. Two strategies were followed: D-PCR, which relies on amplification of DNA directly in blood without DNA extraction using the KAPA Blood PCR Kit, and the C-PCR, which relies on the extraction of bacterial DNA using the Qiagen QiAmp DNA Mini Kit. The following blood samples were included in each strategy: A blood sample with bacterial cerebrospinal meningitis confirmed by blood culture, a normal blood sample seeded with ATCC (13090) reference strain as positive control for the standardization of the PCR procedure, a normal blood sample as a negative control, and an internal negative control test sample (HO). Results Both D-PCR and C-PCR tests gave the expected amplified DNA fragment of 600 bp on agarose gel electrophoresis of both patients’ blood sample and seeded normal blood sample, whereas no amplified products were detected when both tests were performed on normal blood sample or the internal negative HO control. Conclusion Direct blood PCR assay could be a possible easy, rapid, nonexpensive, and specific method for the detection of meningococci in blood samples, particularly in situations in which culture is difficult because of previous treatment, and also could facilitate the large-scale screening of various medical conditions. | ||
| Keywords | ||
| blood sample; conventional PCR; Direct PCR | ||
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