Wang M-Y, Geng J-L, Chen Y-J, Song Y, Sun M, Liu H-Z, Hu C-J
Department of Laboratory Medicine, General Hospital of Jinan Military Region of PLA, Jinan, China.
Department of Central Lab, Weihai Municipal Hospital affiliated to Dalian Medical University, Weihai, China.
Lett Appl Microbiol. 2017 Feb;64(2):138-143. doi: 10.1111/lam.12676.
Methicillin-resistant staphylococci (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 mecA, bla , bla , bla and bla genes from positive blood culture bottles. The technique showed a sensitivity of 10 CFU ml for mecA detection and of 10 CFU ml 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 S. aureus (MRSA), two methicillin-resistant S. epidermidis (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 bla , bla , bla and bla 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.
Many studies on the development of PCR for the detection of resistance genes have already been published, including multiplex PCR methods. However, cross-amplification reactions can be a major concern in multiplex PCR methods. In this study, we developed a highly sensitive and specific multiplex-touchdown PCR assay for simultaneous detection of mecA, bla , bla , bla and bla genes from positive blood culture bottles, cross-amplification was absent and false-positive results were not obtained.
耐甲氧西林葡萄球菌（MRS）和产超广谱β-内酰胺酶（ESBL）细菌是脓毒症中最重要的耐药病原体。在本研究中，开发了一种新的多重降落式PCR方法（MT-PCR），用于快速同时检测阳性血培养瓶中mecA、bla、bla、bla和bla基因的存在。该技术对mecA检测的灵敏度为10 CFU/ml，对其他基因的灵敏度为10 CFU/ml，且在所有基因检测中特异性为100%。在人工接种参考菌株的加标血培养瓶中检测到了所有基因。通过标准微生物学程序，在48小时内从临床血培养标本中分离出3株耐甲氧西林金黄色葡萄球菌（MRSA）、2株耐甲氧西林表皮葡萄球菌（MRSE）和32株产ESBL细菌。通过MT-PCR检测，在4小时内直接从临床血培养标本中的3株MRSA、2株MRSE和29株产ESBL细菌中检测到了相应基因。由于病原体中存在其他ESBL基因型，在另外3瓶产ESBL细菌中未检测到bla、bla、bla和bla基因。同样，所有经培养证明为阴性的瓶子通过PCR检测仍为阴性。所提出的方法快速、灵敏且特异，能够直接从血培养瓶中检测MRS和产ESBL细菌的基因。
关于用于检测耐药基因的PCR技术开发的许多研究已经发表，包括多重PCR方法。然而，交叉扩增反应可能是多重PCR方法中的一个主要问题。在本研究中，我们开发了一种高度灵敏且特异的多重降落式PCR检测方法，用于同时检测阳性血培养瓶中的mecA、bla、bla、bla和bla基因，不存在交叉扩增且未获得假阳性结果。
