金黄色葡萄球菌特异性PCR检测靶点的自动化筛选

Screening of specific target sequences for the PCR detection of Staphylococcus aureus by automatic genomic comparison

摘要:

旨在挖掘用于鉴定金黄色葡萄球菌的高特异性靶点及其PCR检测引物。采用C++语言编程，以金黄色葡萄球菌Staphylococcus aureus MRSA 252基因组编码序列为对象，对2 656个可编码区进行分析，获得特异性靶点序列，并设计PCR扩增引物。对包括葡萄球菌属11个种及其他细菌属在内的共计137株细菌验证引物特异性，筛选获得9个DNA序列，并设计了4对引物。经验证2对引物的特异性较好，其中引物SA3的基因组DNA检测限为13.7 fg/μL，菌体检测限为9.25×102 CFU/mL。结果验证

Abstract:

The aim of this study was to establish a fast and accurate method for developing specific DNA sequences and PCR primers for the detection of Staphylococcus aureus. An automatic C++ program for genomic comparison was used to identify specific DNA sequences from the genome of S. aureus MRSA 252. Four primer pairs were obtained from 9 specific target sequences by comparison of 2656 coding sequences with our local genome database, and 2 pairs of primers were confirmed to be specific to S. aureus by PCR evaluation against 137 bacterial strains, including 11 species of Staphylococcus. Furthermore, the DNA detection sensitivity of primer SA3 was 13.7 fg/μL and the cell sensitivity for this primer was 9.25×102 CFU/mL. This method has overcome the limitations of specific target mining in conventional assays, and it could be easily and widely used for other foodborne pathogens.