The Rapid Nucleic Acid Diagnosis Research For The Detection Of Bacterial Contamination In Platelet

With the increasing demand for platelet products in recent years, the transfusion of platelet concentrates (PCs) had taken the first place of component blood transfusion in the treatment of hematologic diseases. But until recently, the bacterial contamination of platelet concentrates becomes a worldwide concerned problem. Because of the lack of appropriate testing method, the sepsis caused by bacterial contaminations of blood product was considered as one of the most common and serious risks. The culture methods are the conventional method for the detection of bacterial contamination of platelet concentrations in clinic. Although these methods have high sensitivity for most of the bacteria, the complex of operation and the expensive equipment required made these methods only be used in the blood banks or the hospital with good facilities.So our research was focused on the quick PCR (Polymerase chainreaction) at the beginning. After the conserved region of bacterial16S rDNA was amplified, the nucleic acid lateral flow method was used to make the amplicons visible. Based on the research above, we applied the nucleic acid isothermal amplification technology was applied to improve the amplification method for the urgent need of the test the PCs sterility in the field, and this technology was used to detect several kinds of bacterial contamination. The methods and results are as follows:(1) Board range primers and probes were used to amplify and labelled the conserved region of bacterial16S rDNA, and nucleic acid lateral flow dipstick (LFD) to detect the amplicons. This method could detect at least7kinds of bacteria but had no reaction with the human genomic DNA; by using serially diluted standard plasmids, which was constructed by amplifying the conserved region of16S rDNA of Bacillus cereus, the detection limit of PCR-LFD was determined to100copies/PCR which was at least10times higher than that of AGE. The practicability of this16S rDNA PCR-LFD assay for the detection of bacterial contamination in PCs was also valued. The results showed that the detection limit of PCR-LFD for K. pneumonia was5CFU/mL,6.5×104CFU/mL for the S. epidermidis and35CFU/mL for P. aeruginosa. Moreover, the total analysis time of this method was only about2h, which made the PCR based method more suitable for the point of issue required.(2) Through the design of the two pairs of primers, the nicking enzyme mediated amplification (NEMA) method was established, to amplify at least450bp lengthfragment.By the optimization of the parameters (e.g., the concentration of Mg2+, the reaction temperature, the reaction buffer, and the concentration of DMSO et al.) that govern the sensitivity and reproducibility of NEMA, the detection limit of NEMA was boosted as high as100times, but the addition of adjuvant, such as trehalose have no affect on the amplification efficiency. (3) Ey the use of board range primer, the NEMA methodwas applied to detect several kinds of bacteria. After optimization of the parameters, we applied this method was used on the detection of genomic DNA that extracted from real bacteria. The results showed that the NEMA-LFD could detect several contaminated bacteria with the Bacillus cereus at a concentration of1.4×106CFU/test and Klebsiella pneumonia of6.O×107CFU/test.