Study On The Detection Of Group A Streptococcus, Corynebacterium Diphtheria, Legionella Pneumophila By Polymerase Chain Reaction

Objectives The aim of this study is to establish rapid methods for detecting group A streptococcus (GAS), corynebacterium diphtheria and Legionella pneumophila (Lp Respectively by polymerase chain reaction (PCR). This subject's results could contribute to the establishment of multiplex PCR assay and gene chip assay for detecting respiratory communicable diseases.Methods 1. According to the design principle of primers in DNA gene chip assay , pairs of primers are designed by software primer premier 5.0 targeted streptococcal pyrogenic exotoxin B gene ( speB) , diphtheria toxin B gene (toxB) and macrophage infectivity potentiator gene (mip) of Legionella pneumophila (Lp) respectively. The specificity and conservative of these target genes are all verified by "blast" (www.ncbi.nlm.nih.gov) .A routine PCR assay was adopted to amplify the specific DNA fragment of strains tested. 2. A series PCR amplification for differential control strains and DNA samples diluted gradient (1:10) have been used to evaluate the specificity and sensitivity of PCR assay established.Results 1. Detection of GAS by PCR assay: The 345bp specific fragment of speB gene were amplified in all the tested GAS strains including three strains of scarlet fever, whereas it was detected in none of the differential control strains. The lowest limit of detection was 6.5pg genome DNA of GAS strain. 2. Detection of corynebacterium diphtheria by PCR assay: The318bp specific fragment of toxB gene were amplified in all the tested toxigenic corynebacterium diphtheria strains, whereas it was detected in none of the differential control strains. The lowest limit of detection is 850fg/μl genome DNA of corynebacterium diphtheria strain. 3. Detection of Lp by PCR assay: The 340bp specific fragments of mip gene were amplified in all the tested Lp strains, whereas it was detected in none of the differential control strains including three strains of non-pneumophila. The lowest limit of detection is 2.6pg/μl Lp-DNA.Conclusion: 1. These results indicate that the PCR assays based on speB gene, toxB gene and mip gene respectively for detecting group A streptococcus, corynebacterium diphtheria and Legionella pneumophila have high specificity and sensitivity and may be convenient and reliable detection tools for early diagnosis of the three pathogens mentioned above. Due to the limited time we have, the PCR assays established have been verified only by experiment strains. The experiments for appraising the efficacy and accuracy of detecting the clinical sample should be carried out in the future. 2. The design of primers and reaction conditions of PCR in this study keeps to the requests of gene chip assay. So this subject's result could become the basic technique to establish multiplex PCR assay and gene chip assay for detecting respiratory communicable diseases.