多重聚合酶链反应检测耐甲氧西林葡萄球菌

目的 应用一种敏感快速的多重聚合酶链反应（ＰＣＲ）,检测耐甲氧西林的金黄色葡萄球菌（ＭＲＳＡ）和凝固酶阴性葡萄球菌（ＭＲＣＮＳ）。方法 临床分离的北京地区金黄色葡萄球菌１２３株,ＭＲＣＮＳ１２２株,用溶壁素和蛋白酶Ｋ制备模板ＤＮＡ,设计葡萄球菌甲氧西林耐药的决定基因,金黄色葡萄球菌独有一个辅助基因和细菌中均为有１６ＳｒＲＮＡ基在引物,通过多重ＰＣＲ技术对标本进行放增。结果 １２３株金黄色葡萄球菌的     

耐甲氧西林葡萄球菌四种检测方法的比较及临床应用

用ｍｅｃＡ基因检测法、琼脂筛选法、Ｅ试验和Ｋ－Ｂ纸片扩散法检测１００株临床分离的葡萄球菌甲氧西林耐药株（ＭＲＳ）,比较其阳性检出率,对其可靠性和临床实用性进行评估,结果：凡ｍｅｃＡ阳性葡萄球菌（６９／１００）其琼脂筛选法均为阳性,其中６７株苯唑西林ＭＩＣ大于４μｇ／ｍｌ,２株凝固酶阴性葡萄球菌（ＣＮＳ）为２μｇ／ｍｌ。在琼脂筛选法显示耐药的菌株（７３／１００）中,４株为ｍｅｃＡ阴性,且均检出β－内     

耐甲氧西林葡萄球菌耐药性观察

目的研究葡萄球菌的感染及耐药性状况，指导临床用药。方法ＡＰＩ－Ｓｔａｐｈ鉴定分离菌株，药敏试验应用Ｋ－Ｂ法、最低抑菌浓度（ＭＩＣ）法、琼脂筛选法及ｍｅｃＡ基因聚合酶链反应（ＰＣＲ）检测耐甲氧西林葡萄球菌（ＭＲＳ）；并分别测定了ＭＲＳ、甲氧西林敏感葡萄球菌（ＭＳＳ）对青霉素等１３种抗生素的耐药率（Ｋ－Ｂ法）；分析了病人住院时间、抗生素使用与ＭＲＳ分离率间的关系。结果３０１株葡萄球菌中，耐甲氧西林金黄色葡萄球菌（ＭＲＳＡ）占４４．０％（３７／８４）（筛选法），耐甲氧西林凝固酶阴性葡萄球菌占６１．８％（１３４／２１７）（筛选法），ｍｅｃＡ基因ＰＣＲ法、琼脂筛选法检测ＭＲＳ阳性率较高；ＭＲＳ对青霉素等１３种抗生素的耐药率均高于ＭＳＳ；病人住院天数、抗生素使用与ＭＲＳ的分离率呈正相关。结论ＭＲＳ感染较为严重，需加强其对抗生素包括万古霉素的耐药性监测；注意ＭＲＳ检测方法存在的问题。     

耐甲氧西林葡萄球菌的临床检测

目的探讨沈阳地区耐甲氧西林葡萄球菌（ＭＲＳ）的感染情况，流行特征，应用ＭＲＳ鉴别培养，聚合酶链反应产和的酶联检测（ＥＤ－ＰＣＲ）及常规药敏试验法。结果从７５株葡萄球菌中同耐甲氧西林金黄色葡萄球菌（ＭＲＳＡ）１０株，耐甲氧西林血浆凝固酶阴性力葡萄球菌（ＭＲＣＮＳ）１３株。１０株ＭＲＳＡ的血浆凝固酶型别主要集中在Ⅳ型，Ⅱ型次之，结论 沈阳地区耐甲氧西林葡萄球菌染较为严重（２３／７６），主要的血浆凝固酶     

耐甲氧西林葡萄球菌的临床检测

目的探讨沈阳地区耐甲氧西林葡萄球菌（ＭＲＳ）的感染情况、流行特征。方法应用ＭＲＳ鉴别培养，聚合酶链反应产物的酶联检测（ＥＤ－ＰＣＲ）及常规药敏试验法。结果从７６株葡萄球菌中检出耐甲氧西林金黄色葡萄球菌（ＭＲＳＡ）１０株，耐甲氧西林血浆凝固酶阴性葡萄球菌（ＭＲ－ＣＮＳ）１３株。１０株ＭＲＳＡ的血泺凝固酶型别主要集中在Ⅵ型，Ⅱ型次之。结论沈阳地区耐甲氧西林葡萄球菌感染较为严重（２３／７６），主要的血浆凝固酶型别为Ⅲ型。３种检测方法比较，ＥＤ－ＰＣＲ法具有操作简便、特异、敏感等优点，宜于推广应用     

金黄色葡萄球菌耐药性分析

目的了解金黄色葡萄球菌（ＳＡ）尤其是耐甲氧西林金黄色葡萄球菌（ＭＲＳＡ）的耐药状况，指导临床合理用药。方法对本院感染标本中分离出的２２３株ＳＡ分别进行了药敏试验和β－内酰胺酶测试，并以“ＷＨＯＮＥＴ３”软件对试验数据进行分析处理。结果ＭＲＳＡ占ＳＡ感染标本总数的５８．３％，ＭＲＳＡ及甲氧西林敏感金黄色葡萄球菌（ＭＳＳＡ）产β－内酰胺酶的百分率无明显差异。ＭＲＳＡ与ＭＳＳＡ皆对万古霉素敏感。此外，ＭＲＳＡ对１８种抗生素中的１５种呈现多重耐药，耐药率介于２８％～１００％。而多数ＭＳＳＡ仅对西林Ｇ和氨苄西林耐药。结论万古霉素是治疗ＭＲＳＡ感染的首选抗生素。ＭＲＳＡ的耐药性应引起广泛关注。     

耐甲氧西林葡萄球菌耐药性观察

目的 研究葡萄球菌的感染及耐药性状况，指导临床用药。方法 ＡＰＩ－Ｓｔａｐｈ鉴定分离菌株，药敏试验应用Ｋ－Ｂ法、最低抑菌浓度（ＭＩＣ）法、琼脂筛选法及ｍｅｃＡ基因聚合酶链反应（ＰＣＲ）检测耐甲氧西林葡萄球菌（ＭＲＳ）；并分别了ＭＲＳ、甲氧西林敏感葡萄球菌（ＭＳＳ）对青霉素等１３种抗生素的耐药率（Ｋ－Ｂ法）；分析了病人住院时间、抗生素使用与ＭＲＳ分离率间的关系。结果 ３０１株葡萄球菌中，耐甲西林金黄     

多重PCR在幽门螺杆菌检测及分型中的应用

应用多重ＰＣＲ同时扩增１６ＳｒＲＮＡ和ｃａｇＡ基因来检测幽门螺杆菌感染及其分型。ＰＣＲ产物经ＤＮＡ序列测定证实为转异性扩增，敏感度为１０＾２ＣＦＵ／ｍｌ。４８株Ｈｐ菌株中，Ｉ型菌株（ｃａｇＡ＋）２６株（５４．２％）；５５０份胃粘液标本，Ｈｐ阳性（１６ＳｒＲＮＡ基因＋）２１６份（４７．５％），其中Ｉ型Ｈｐ１３９份（５３．３％）。     

凝固酶阴性葡萄球菌苯唑西林筛选试验药物浓度的探讨

目的 选择一合适的筛选法检测凝固酶阴性葡萄球菌（ＣＮＳ）和苯唑西林（ＯＸＡ）浓度。方法 通过Ｋ－Ｂ纸片法、琼脂稀释法和β－内酰胺酶抑制试验对１４１株ＣＮＳ进行检测。结果 选择ＯＸＡ０．２５μｇ／ｍｌ时，有８株ＯＸＡ敏感株（ＭＳＳ）被误判为耐药株（ＭＲＳ），误判率为８．３３％；选择ＯＸＡ０．５μｇ／ｍｌ时，１株ＭＳＳ误判为ＭＲＳ，误判率为１．０４％；选择ＯＸＡ０．７５μｇ／ｍｌ时无ＭＳＳ或ＭＲＳ的误     

快速检测耐甲氧西林金黄色葡萄球菌方法的应用评价

快速、准确地筛选高危病人成为医院有效控制耐甲氧西林金黄色葡萄球菌 (ＭＲＳＡ)的关键。目前 ,临床上对ｍａｃＡ基因以及其编码ＰＢＰ2ａ进行检测是最有效的方法。我们将ＰＢＰ2ａ胶乳法和ｍｅｃＡ基因探针法与ＰＣＲ检测法进行了比较 ,并评价了它们的可靠性和临床应用价值 ,报道如下。一、材料与方法1 菌株 :采用ＡＰＩ系统试条鉴定为金黄色葡萄球菌 ,共96株。分别选ＡＴＣＣ4330 0、ＡＴＣＣ2 5 92 3为阳性和阴性质控菌。2 ＭＲＳＡ筛选 :按标准制备菌悬液 ,接种于苯唑西林琼脂筛选平板上 ;35℃ ,2 4ｈ孵育 ,有菌落生长为ＭＲＳＡ。3 …     

A study of the regulating gene of femA from methicillin-resistant Staphylococcus aureus clinical isolates

The regulating gene of femA was studied in methicillin-resistant Staphylococcus aureus (MRSA). High-level MRSA, low-level MRSA and methicillin-sensitive S. aureus (MSSA) were identified by agar diffusion. Beta-lactamases were detected by nitrocephin and the presence of the mecA gene was determined by polymerase chain reaction (PCR). Only isolates that were both beta-lactamase-negative and mecA-positive were used. The femA gene and its 250 base pair (bp) upstream sequence were amplified by PCR and expression was determined by real-time fluorescent quantitative PCR. The 250 bp upstream sequence was labelled by BrightStar Psoralen-Biotin and detected by electrophoretic mobility shift assay (EMSA). Expression levels of femA in MSSA, low-level MRSA and high-level MRSA were 3.53 x 10(-3)% - 29.91%, 5.54 x 10(-3)% - 3.1 x 10(2)% and 13.88 - 5.50 x 10(4)%, respectively. EMSA detected a signal shift in 57 high-level MRSA isolates but not in four low-level MRSA and four MSSA strains. Expression of femA in high-level MRSA (non-beta-lactamase-producing) was higher than in low-level MRSA and MSSA. The femA regulating gene probably lies in the 250 bp upstream sequence in MRSA and high-level expression is essential for high-level methicillin resistance.     

多重PCR检测临床分离金黄色葡萄球菌lukS/F-PV和mecA基因

目的建立一种新的多重PCR方法,检测临床分离金葡菌的杀白细胞素毒力基因(lukS/F-PV)和甲氧西林耐药基因(mecA)。方法收集我院2006年1—12月从临床多种标本中分离鉴定的不重复金葡菌,应用多重PCR技术,优化反应条件,同时扩增葡萄球菌属特异性基因16S rRNA、金葡菌种特异性基因nuc、lukS/F-PV基因和mecA基因,扩增产物经凝胶成像系统分析。结果217株金葡菌经多重PCR检测,31株是16S rRNA+nuc+lukS/F-PV+mecA基因型,3株是16SrRNA+nuc+lukS/F-PV基因型,135株是16S rRNA+nuc+mecA基因型,40株是16S rRNA+nuc基因型,其中5株仅扩增出16S rRNA基因,3株仅扩增出nuc基因。lukS/F-PV基因和mecA基因测序显示与GenBank中的已有序列具有高度同源性,其阳性率分别为15.7%(34/217)和76.5%(166/217);34株lukS/F-PV基因阳性的分离株中,31株为MRSA,占91.2%(31/34)。结论本方法适用于快速检测和鉴定产杀白细胞素MRSA,lukS/F-PV基因阳性的菌株以MR...     

Detection of ileS-2 gene encoding mupirocin resistance in methicillin-resistant Staphylococcus aureus by multiplex PCR.

The presence of the ileS-2 gene, responsible for mupirocin resistance, in clinical isolates of methicillin-resistant Staphylococcus aureus was determined by multiplex polymerase chain reaction. Three pairs of primers were used, which yielded specific fragments of femA (encoding a unique feature of S. aureus), mecA (encoding resistance to methicillin) and ileS-2 genes. The multiplex polymerase chain reaction system is an easy and time-saving technique that, together with a rapid method for DNA extraction by boiling, may be incorporated as a routine analysis in clinical diagnostic laboratories.     

应用DNA探针检测甲氧西林耐药葡萄球菌

目的建立一种快速检测葡萄球菌和甲氧西林耐药葡萄球菌的斑点杂交技术。方法设计金黄色葡萄球菌nuc基因、甲氧西林耐药mecA基因、葡萄球菌tuf基因的特异引物,用聚合酶链反应合成其特异DNA探针,并用生物素标记,分别与固定在硝酸纤维素膜上的标准菌株和临床分离株模板DNA杂交,观察其敏感性和特异性。结果3对引物分别扩增出270bp、310bp、370bp3种DNA探针,均具有高度特异性。50株金黄色葡萄球菌tuf、nuc基因均为阳性：mecA基因阳性者22株。30株表皮葡萄球菌tuf基因均为阳性,nuc基因均为阴性,mecA基因阳性者9株。而其他非葡萄球菌与3种DNA探针杂交结果均为阴性。该方法可检测出1 ng细菌DNA。结论斑点杂交技术检测耐甲氧西林葡萄球菌快速、有效,具有较高的应用价值。     

BacTAlert240全自动血培养仪病原菌分布与药敏分析

目的 了解血培养阳性常见病原菌分布及其耐药情况,为临床正确选用抗生素提供依据.方法 用BacT/Alert 240全自动血培养仪对4458份血液标本进行检测,用VITEK-32鉴定系统对细菌进行鉴定,药敏试验采取K-B纸片扩散法按CLSI 规定的标准进行.结果 从4458份血培养中共检出430株病原菌,阳性率为8.8%.主要致病菌有凝固酶阴性葡萄球菌、大肠埃希菌、金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌5种,占菌株总检出率59%,其中耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）占凝固酶阴性葡萄球菌的70.8%,耐甲氧西林金黄色葡萄球菌（MRSA）占金黄色葡萄球菌的43.2%,未发现耐万古霉素的葡萄球菌.大肠埃希菌和肺炎克雷伯菌产超广谱β-内酰胺酶（ESBLs）的菌株分别为46.7%和28.3%.结论 血培养中病原菌复杂多样,耐药率高及时检测血液中病原菌的耐药性对于指导临床合理用药,防止滥用抗生素减少耐药菌株的产生是十分必要的.     

Rapid detection of mecA in methicillin resistant Staphylococcus aureus using cycling probe technology.

A novel method has been developed for the detection of the mecA gene, that confers the principle mechanism of methicillin resistance in staphylococci. Cycling Probe Technology (CPT) is a rapid, simple, isothermal method for the detection of specific target sequences. CPT utilizes a unique chimeric DNA-RNA-DNA probe sequence that provides an RNase H sensitive scissile link when hybridized to a complementary target DNA sequence. In the presence of target DNA, the cycling reaction converts full-length chimeric probe into cleaved probe fragments, which accumulate and are quantified. A cycling probe designed for detection of a specific sequence within the mecA gene was used to develop a culture confirmation assay for methicillin resistant Staphylococcus aureus. The CPT assay was used to screen 238 S. aureus isolates and the results were in complete agreement with detection of the mecA gene by polymerase chain reaction (PCR). Detection of mecA should be considered the gold standard for determining methicillin resistance in S. aureus. This study demonstrates the feasibility of using CPT to meet this need.     

Survey of the methicillin resistance-associated genes mecA, mecR1-mecI, and femA-femB in clinical isolates of methicillin-resistant Staphylococcus aureus.

The restriction site polymorphism of the chromosomal femAB region and the first appearance of the regulatory element mecR1-mecI associated with the methicillin resistance determinant (mec) were analyzed in 192 initially methicillin resistant (Mcr) Staphylococcus aureus clinical isolates collected between 1965 and 1990 in the Zurich area. Forty-three of the strains lost the resistance spontaneously. All isolates that were still Mcr hybridized with mecA, the gene for the low-affinity penicillin-binding protein PBP 2'. Mcr strains isolated before 1977 lacked sequences that hybridized with mecR1-mecI, a regulatory element controlling the expression of mecA; exceptions to this were one strain isolated in 1966 and one strain isolated in 1972. The size of the EcoRV fragment carrying femA, a chromosomally encoded factor involved in pentaglycine side chain formation of the peptidoglycan and essential for the expression of methicillin resistance, was conserved in all strains but one, which was susceptible to methicillin even though it carried a functional mecA gene. The methicillin susceptibility of this particular strain was presumably due to a spontaneous femA-like mutation. The 192 strains belonged to seven different EcoRV restriction fragment patterns recognizable with a 10.5-kb probe covering the femAB region. Some 93% of the 149 Mcr strains belonged to pattern A, and the remaining Mcr strains shared patterns A' and B. The 42 isolates which spontaneously lost their resistance upon storage and revival represented all seven different patterns. This strong conservation of femA suggests an important role for femA in cell wall metabolism and methicillin resistance.     

Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction

A multiplex real-time polymerase chain reaction (RT-PCR) targeting the mecA and nuc genes was developed for the detection of methicillin resistance and identification of Staphylococcus aureus. Novel mecA and nuc primers and fluorescence resonance energy transfer hybridization probes specific for the mecA and nuc genes were evaluated. The assay was performed using the LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) and evaluated against the traditional gel-based multiplex PCR (PCR-gel) method currently used at Royal Perth Hospital. Clinical isolates (n = 222) and isolates from a culture collection library (n = 206) were tested by both assays in parallel. The RT-PCR assay was 100% sensitive and specific for the detection of methicillin resistance and for the identification of S. aureus when compared with the PCR-gel assay. Results from the RT-PCR assay showed 5 isolates with lower efficiency fluorescence curves for the nuc gene PCR fragment. DNA sequencing showed mutations within the region of the probe-binding sites compared with the reference strain. The results of the RT-PCR assay were available within 2 h. This rapid mecA/nuc RT-PCR assay is a suitable and practical tool for the routine detection of methicillin resistance and identification of S. aureus, which can be easily incorporated into the diagnostic molecular microbiology laboratory work flow.     

三种检测耐甲氧西林金黄色葡萄球菌方法的比较

[摘要]目的:对耐甲氧西林金黄色葡萄球菌(MRSA)3种检测方法的可靠性和临床实用性进行评价。方法：用检测mecA基因的PCR扩增法、苯唑西林纸片扩散法、头孢西丁制片扩散法对临床分离的84株金黄色葡萄球菌进行检测并作比较。结果:苯唑西林纸片扩散法检的敏感性、特异性分别为97.6%、95.3%,mecA基因的PCR扩增法和头孢西丁纸片扩散法的敏感性、特异性均为100.0%。结论:PCR技术检测MRSA具有快速、敏感和特异的特点,是一种非常有效的鉴别方法。纸片扩散法简便易行,成本低廉, 尤其是头孢西丁纸片扩散法的敏感性和特异性较苯唑西林纸片扩散法更高,值得在临床实验室中推广应用。 [关键词]　耐甲氧西林金黄色葡萄球菌；mecA基因；PCR；苯唑西林；头孢西丁     

Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. The ESPRIT Trial

Multiresistant staphylococci (82 Staphylococcus aureus and 114 coagulase-negative staphylococci) were characterized by testing with rapid multiplex polymerase chain reaction (PCR) assays for species identification and detection of associated antibiotic resistance genes. These 196 staphylococci were isolated from 149 adult patients who developed wound infection after elective coronary artery bypass grafts and/or valve surgery. The multiplex PCR assays allowed identification of the most common staphylococcal species with S. aureus- and Staphylococcus epidermidis-specific primers as well as the detection of the erythromycin resistance genes ermA, ermB, ermC and msrA, the aminoglycoside resistance gene aac(6')-aph(2"), the oxacillin resistance gene mecA and the penicillin resistance gene blaZ. There was a very good correlation between the genotypic analysis by PCR and the phenotype determined by standard methods of susceptibility testing and identification of staphylococcal species: 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin resistance and 100% for S. aureus and S. epidermidis species identification. This study suggests that the incidence and distribution of the tested clinically relevant antibiotic resistance genes in staphylococci associated with infections after cardiac surgery do not differ from those in strains from other infections. These multiplex PCR assays may be used as diagnostic tools to replace or complement standard methods of susceptibility testing and identification of staphylococci.