Assessment of 16s rRNA and Multiplex Real-Time PCR methods for the detection of some pathogenic bacteria in symptomatic septic patients
SUMMARY
There has been great interest in the development of new diagnostic methods able to speed up the process of determining presence of infection and identification of bacterial pathogens in patients with suspected sepsis. Therefore, designing a Multiplex Real Time PCR (qPCR) method can detect and identify any bacterial DNA present in small amounts of patient´s blood samples.
In this study a total of 100 blood samples were collected from 30 patients with diagnosed cancer from Nanakali Hospital and 70 patients from Intensive Care Unit (ICU) from PAR Private Hospital during January-March of 2016. Total Genomic DNA was directly extracted from blood samples using a bacteria-specific DNA isolation kit. 16s rRNA universal primers unique for all Eubacteria used to identify the presence of any bacteria in the samples. Moreover, two separate Multiplex qPCRs were conducted for simultaneous detection of pathogenic bacteria by selecting gene-specific primers including Ply gene of Streptococcus pneumonia, Sa442 gene of Staphylococcus aureus for gram positives, and Efp gene of Acinetobacter spp., uidA of E. coli and ntrA gene of Klebsiella pneumonia for gram negative bacteria.
Overall primers resulted in specific and successful amplification and detection of their corresponding genes from each bacteria. The melt peaks of PCR amplicons from Streptococcus pneumoniae and Staphylococcus aureus resulted in no significant differences (SD) with the Tm of standard positive controls (P values 0.17 and 0.54) revealing Tm of 84.31±0.06 and 79.43±0.23 (Mean ±SE) respectively. While for the gram negative bacteria, the Tm of E. coli, K. pneumoniae, and Acinetobacter spp. PCR amplicons from samples revealed no SD with their corresponding standard positive controls having P values of 0.19, 0.12, and 0.12 and average Tm of 87.7±0.11, 88.27±0.13 and 84.25±0.55 (Mean ± SE) respectively. Moreover, bacteremia due to a single organism was found in 25 cases (86.2%), and that due to more than one organism was found in 4 (13.7%) samples, and the prevalence of the most dominant bacteria that causes Blood stream infection in the tested samples in Erbil was Streptococcus pneumoniae, while Escherichia coli was the second most prevalent.
Finally, the 16s rRNA primer was able to identify each bacterium included in this study and the qPCR assay reveled a sensetivity limit of the quantification between 1.32×10-3 to 11.1×105 cells/ml which means the lowest number of bacteria that can be quantified with this method was 1.32×10-3 and the highest number of bacteria that can be quantified was 11.1×105 cells/ml.
posted: 21/05/2017