Selective amplification of bacterial 16S rDNA sequence from clinical samples

Abstract

Background: Conventional blood culture method is time consuming and less sensitive; when fastidious or un-culturalable organisms are involved. The use of PCR targeting the 16S rRNA allows detection of bacteria; however, these primers have ability to co-amplify human DNA. This Polymerase Chain Reaction (PCR) method is based on nucleic acid amplification test.

Objective of the study: This study determined a method for selective amplification of bacterial DNA from clinical samples without co-amplification of human DNA.

Materials and methods: Seventy one blood samples from clinically suspected cases of early onset neonatal sepsis were collected and analysed in parallel by culture and 16S rRNA amplification. DNA was extracted using commercial extraction QiAmp mini DNA kit and subjected to 16S rRNA amplification. The products were sequenced, analysed and compared with blood culture results. Positive and negative controls were used for extraction and amplification respectively.

Results: Out of 71 samples analysed, 5 (7.0%) samples by blood culture were equally positive for 16S rRNA PCR; the PCR was also able to identified 16 (22.5%) more positive samples which blood culture could not identify, but only 1 (1.4%) sample was identified positive using blood culture while PCR identified it as negative. During the study, 7 (9.9%) samples were identified positive by conventional blood culture but later found to be contaminants.

Conclusion: This study confirmed the presence of 16S rRNA among bacterial isolates and modification of PCR protocol with shorter denaturation temperature and time, leading to selective amplification of bacterial DNA. Therefore, there is need to carry-out this investigation on both culturable and unculturable specimens.

Keywords:16SrRNA amplification; bacterial DNA; human DNA and Polymerase chain