Direct detection of m>mecAm>,m> blam>_SHVm>, blam>_CTX-M, m>blam>_TEM and m>blam>_OXA genes from positive blood culture bottles by multiplex-touchdown PCR assay

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• 作者：M.-Y. Wang ; J.-L. Geng ; Y.-J. Chen ; Y. Song ; M. Sun ; H.-Z. Liu and C.-J. Hu
• 刊名：Letters in Applied Microbiology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：64
• 期：2
• 页码：138-143
• 全文大小：298K
• ISSN：1472-765X

文摘

Methicillin-resistant m>staphylococcim> (MRS) and ESBL(Extended-Spectrum β-Lactamase)-producing bacteria are the most important resistant pathogens in sepsis. In this study, a new multiplex-touchdown PCR method (MT-PCR) was developed to detect rapidly and simultaneously the presence of m>mecAm>,m> blam>_SHVm>, blam>_CTX-M, m>blam>_TEM and m>blam>_OXA genes from positive blood culture bottles. The technique showed a sensitivity of 10³ CFU ml^−1 for m>mecAm> detection and of 10² CFU ml^−1 for other genes, and 100% specificity in the detection of all genes. All genes were detected in the spiked blood culture bottles artificially contaminated with reference strains. Three methicillin-resistant m>S. aureusm> (MRSA), two methicillin-resistant m>S. epidermidism> (MRSE) and 32 ESBL-producing bacteria, were isolated from the clinical blood culture specimens in 48 h by standard microbiological procedures. The corresponding genes were detected directly in the three MRSA, two MRSE and 29 ESBL-producing bacteria from the clinical blood culture specimens in 4 h by MT-PCR assay. None of the m>blam>_SHV,m> blam>_CTX-M, m>blam>_TEM and m>blam>_OXA genes were detected in three other bottles with ESBL-producing bacteria because of other ESBL genotypes in the pathogens. Likewise, all bottles proven negative by culture remained negative by PCR. The proposed method was rapid, sensitive and specific, and was able to directly detect the genes of MRS and ESBL-producing bacteria from the blood culture bottles.