6种食品致病菌的多重PCR检测

目的寻求食物中毒诊断及食品检测的有效方法。方法建立多重PCR体系一次检测多种食品致病菌方法。结果可以一次检测出肠毒性大肠埃希菌(E.coli-ETEC),蜡样芽胞杆菌(Bacilluscereus),沙门菌属(Salmonellaspp),大肠埃希菌O157:H7(E.coli-O157:H7),志贺菌属(Shigellaspp),霍乱弧菌(Vibriocholerae)等菌属或菌。结论多重PCR体系检测6种菌DNA的灵敏度最低为10.30fg,与单独的PCR检测的灵敏度相同。将它做成体外基因诊断试剂盒,将成为细菌性食物中毒诊断及食品检测的有效工具。     

Multiplex PCR for detection of Enterobacteriaceae in blood

BACKGROUND: Rapid and sensitive methods are needed to detect the small numbers of bacteria that may sometimes contaminate units of blood during collection. A multiplex 5'-nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood. STUDY DESIGN AND METHODS: Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia enterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probes were designed: SL-1 detected Serratia, Klebsiella, and Enterobacter species, and YE-3 detected Y. enterocolitica. RESULTS: When TaqMan PCR was performed with chromosomal DNA isolated from pure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of detection with probe SL-1 was 1 to 2 CFUs. For S. marcescens, the sensitivity was 8 CFUs. The limit of detection for Y. enterocolitica with probe YE-3 was 2 CFUs. When total chromosomal DNA was extracted from whole-blood samples spiked with different numbers of Y. enterocolitica, S. liquefaciens, E. cloacae, or K. pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood. CONCLUSION: The 5'-nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.     

Multiplex PCR for detection of acquired carbapenemase genes

A rapid and reliable PCR-based technique was developed for detection of genes encoding carbapenemases belonging to different classes. Primers were designed to amplify the following 11 genes: bla(IMP), bla(VIM), bla(NDM), bla(SPM), bla(AIM), bla(DIM), bla(GIM), bla(SIM)bla(KPC), bla(BIC), and bla(OXA-48). Three different multiplex reaction mixtures were defined and evaluated for the detection of all these 11 genes. Using optimized conditions, each reaction mixture allowed to identify the respective genes, with PCR giving distinct amplicon sizes corresponding to the different genes for each mixture. We reported here a rapid and reliable technique for screening all clinically relevant carbapenemase genes.     

Multiplex real-time SYBR Green I PCR assays for simultaneous detection of 15 common enteric pathogens in stool samples

Diarrheal diseases account for more than 50% of foodborne diseases worldwide, the majority of which occur in infants and young children. The traditional bacterial detection method is complex and time-consuming; therefore, it is necessary to establish a rapid and convenient detection method that can detect multiple pathogens simultaneously. In this study, we developed a set of five multiplex real-time SYBR Green I PCR assays to simultaneously detect 15 common enteric pathogens based on the Homo-Tag Assisted Non-Dimer system. These assays effectively reduced primer-dimer formation and improved the stability, uniformity, and amplification efficiency of multiplex PCR. The detection limit of the multiplex SYBR Green I PCR system was approximately 10⁴–10⁶ CFU/mL for stool specimens. Furthermore, we vitrified heat-unstable components on the cap of a reaction tube, showing that Taq DNA polymerase, dNTPs, primers, and SYBR Green I remained stable at 25 °C. In summary, we developed multiplex SYBR Green I PCR assays that can simultaneously detect 15 enteric pathogens. This method is comprehensive, rapid, inexpensive, accurate, and simple and displays high specificity.     

Multiplex PCR using real time DNA amplification for the rapid detection and quantitation of HTLV I or II

A multiplex 'real-time' polymerase chain reaction (PCR) has been established as a general technique for the quantitation of proviral human T-lymphotrophic virus types 1 and 2 (HTLV-I/II). The technology utilizes fluorescence to measure amplification products from the tax gene of Human T-cell lymphotropic virus type 1 or the 5' long terminal repeat of Human T-cell lymphotropic virus type 2. The quantitative amplification of the standard was linear across four orders of magnitude with nearly identical amplification efficiencies for monoplex or the biplex format from 1.4 copes/assay (60 copies proviral DNA/0.5 micrograms human DNA) to 6000 copies/assay (240000 proviral copies/0.5 micrograms human DNA). The human beta-globin gene was used to normalize for human DNA input to determine the proviral DNA load. Three hundred fifty-six specimens received by Specialty Laboratories for HTLV I/II detection provided identical results in the detection of HTLV I/II proviral DNA. No additional positive specimens were identified with the biplex assay format. The coefficient of variation for the proviral DNA load was less than 30% for HTLV I or II quantitation (n=5). For spiked specimens, two groups of five separate 0.25 ml blood specimens (20 total) were spiked, respectively, with 0, 9.6, 48, 240 and 1200 copies of HTLV I or HTLV II DNA standards. The specimens were amplified with the HTLV I/II multiplex format. Twenty of twenty expected negative HTLV I or HTLV II specimens were negative (100% specificity) and 14/16 specimens spiked with 48 copies or more HTLV I were detected (87.5% sensitivity). Thirteen of sixteen HTLV II spiked specimens (>48 copies of HTLV II standard per 10 assays) were detected (81.2%). The real-time detection provides accurate and reliable results in a single amplification for both HTLV (I or II) targets with a more rapid turnaround time and a decrease in material required for results.     

Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens

A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16–18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.     

细菌革兰双检荧光定量PCR方法检测新生儿败血症

目的 建立细菌革兰阴、阳性菌双重实时荧光定量检测体系,探讨其检测败血症的临床应用价值.方法 分析细菌16SrRNA基因序列,在高度保守区自行设计通用引物和革兰阴性和阳性分型探针,选取临床较常见的35株菌株进行细菌革兰双检实时荧光定量PCR方法检测;对临床疑为败血症的512例新生患儿分别做细菌革兰双检荧光定量PCR和血培养检测.结果 细菌革兰双检荧光定量PCR具有较好的敏感性和特异性,能稳定检测到10 CFU左右细菌数.35株菌株进行细菌革兰双检荧光定量PCR检测,均为阳性,且革兰阴、阳性菌能正确分型和定量.巨细胞病毒、EB病毒、乙肝病毒、新型隐球菌及白色念珠菌、人基因组DNA及空白对照均为阴性.对临床疑为败血症的512份新生患儿标本中,革兰双检荧光定量PCR检测血标本阳性率8.20%(42/512),血培养阳性率6.25%(32/512),前者明显高于后者,差异具有统计学意义(χ<'2>=8.10,P<0.01),30例非感染性疾病同期患儿血标本革兰双检荧光定量PER及细菌培养均为阴性.若以血培养作为对照,细菌革兰双检荧光定量PCR方法的诊断敏感度为100%,特异度为97.92%,准确性98.05%.结论 建立了用通用引物和分型双荧光探针的细菌革兰双检荧光定量PER方法.其检测快速、准确,具有很大的临床推广价值.     

Multiplex PCR for detection of aminoglycoside resistance genes in enterococci

A multiplex PCR procedure for detecting the aminoglycoside resistance genes aac(6')-Ie-aph(2")-Ia, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aph(3')-IIIa, and ant(4')-Ia was evaluated and found to determine accurately the presence of these genes in enterococci.     

Rapid detection of eight causative pathogens for the diagnosis of bacterial meningitis by real-time PCR

We aimed to detect causative pathogens in cerebrospinal fluid (CSF) collected from patients diagnosed with bacterial meningitis by real-time polymerase chain reaction (PCR). In addition to Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae described previously, five other pathogens, Neisseria meningitidis, Escherichia coli, Streptococcus agalactiae, Staphylococcus aureus, and Listeria monocytogenes, were targeted, based on a large-scale surveillance in Japan. Results in CSF from neonates and children (n = 150), and from adults (n = 18) analyzed by real-time PCR with molecular beacon probes were compared with those of conventional culturing. The total time from DNA extraction from CSF to PCR analysis was 1.5 h. The limit of detection for these pathogens ranged from 5 copies to 28 copies per tube. Nonspecific positive reactions were not recognized for 37 microorganisms in clinical isolates as a negative control. The pathogens were detected in 72.0% of the samples by real-time PCR, but in only 48.2% by culture, although the microorganisms were completely concordant. With the real-time PCR, the detection rate of H. influenzae from CSF was high, at 45.2%, followed by S. pneumoniae (21.4%), S. agalactiae (2.4%), E. coli (1.8%), L. monocytogenes (0.6%), and M. pneumoniae (0.6%). The detection rate with PCR was significantly better than that with cultures in patients with antibiotic administration (χ² = 18.3182; P = 0.0000). In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.     

Multiplex PCR for the detection of tetracycline resistant genes

Specific primer pairs were selected for the PCR amplification of 14 tetracycline resistant genes commonly found in Gram positive and Gram negative organisms. Combinations of primer pairs were used in multiplex PCR reactions to detect specific groups of tet genes as follows; Group I tet (B), tet (C), tet (D); Group II tet (A), tet (E), tet (G); Group III tet (K), tet (L), tet (M), tet (O), tet (S); Group IV tetA (P), tet (Q), tet (X). To test the multiplex PCR, Groups I and II were used on 25 clinical isolates of Salmonella enterica serovar Typhimurium DT104. Group III primers were used to investigate 19 clinical isolates of methicillin-resistant Staphylococcus aureus. Multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains when compared with using an individual PCR for targeting each gene. It may also be a useful method to differentiate the types of tetracycline resistance when used as an additional marker for the purpose of outbreak investigation and surveillance.     

Multiplex detection of antibiotic resistance genes using padlock probes

The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10⁴ DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 10⁷ cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.     

The role of a real-time PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples

The rapid diagnosis of pathogens and prompt initiation of appropriate antibiotic therapy are critical factors to reduce the morbidity and mortality associated with sepsis. In this study, we evaluated a multiplex polymerase chain reaction (PCR-M) test that detects bacteria and fungi in whole-blood specimens, comparing its features to those of a blood culture (BC). Following evaluation of the performance for sensitivity and specificity of PCR-M, 78 blood samples from 54 patients with suspected bacterial infections were evaluated. Whole-blood samples for PCR-M were collected at the same time as BC, and PCR-M results were compared with BC results. As a result, minimum sensitivity of the kit was 1–100 cfu/ml. The PCR-M test correctly identified specificity for 13 out of 14 strains blinded to the assay analyst. Of 78 blood samples examined, 56 (72%) were negative by both methods, and 22 (28%) were positive by at least one of the two methods. PCR-M detected organisms in 21 cases (27%) compared with 12 cases (15%) in BC. The correlation of positives between PCR-M and BC was 92% (11/12), and both methods identified the same organisms in these 11 cases. With higher positive rate compared with BC, PCR-M could detect and identify potentially significant microorganisms within a few hours by using a small volume of a single whole-blood sample. Early detection of microorganisms has the potential to facilitate early determination of appropriate treatment and antimicrobial selection.     

Multiplex PCR for simultaneous detection of macrolide and tetracycline resistance determinants in streptococci

Resistance to macrolides and tetracyclines is increasing among streptococci and co-occurs as their resistance determinants are carried on the same mobile element. We developed a multiplex PCR to facilitate simultaneous and specific detection of resistance determinants for both macrolides [erm(A), erm(B), and mef(A/E)] and tetracyclines [tet(M), tet(O), tet(K), and tet(L)] in streptococci.     

性病病原体多重聚合酶链反应检测方法的建立和验证

目的建立同时检测淋球菌、沙眼衣原体、解脲支原体和单纯疱疹病毒-2致病微生物的多重聚合酶链反应（PCR）方法,用于四种性病的诊断和治疗监测。方法根据淋球菌外膜孔蛋白基因、沙眼衣原体基因、解脲支原体尿素酶基因和单纯疱疹病毒-2的DNA聚合酶基因序列,各设计一对特异性引物,优化反应体系和条件进行多重PCR反应。结果4对引物分别扩增出327、167、426、391bp的目的条带,并且特异性强,与其他非目的病原体基因不发生反应。结论本研究建立的多重PCR方法为相关病原体的快速检测提供方法。     

Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing.     

Multiplex PCR for rapid detection of genes encoding class a carbapenemases

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.     

三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应(PCR)方法。方法本研究依据沙门菌侵袭基因正调节蛋白(hilA)基因、变形杆菌溶血素(hpmA)基因和金黄色葡萄球菌特异性pSa-442序列,运用Primer Premier 5.0分别设计3对特异性引物,预计PCR扩增的目的基因片段分别为为580bp、401 bp、256 bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16(43)正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为:94.07 pg沙门菌DNA,140.85 ng变形杆菌DNA,1.41 ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌,4 h培养后样品的最低检测限度分别为:沙门菌100菌落形成单位(CFU)/mL、变形杆菌101CFU/mL、金黄色葡萄球菌100CFU/mL。结论该方法特异性和灵敏度高,检验周期短,可用于对食品中多种致病菌的快速诊检和监控。     

三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应（PCR）方法。方法本研究依据沙门菌侵袭基因正调节蛋白（hilA）基因、变形杆菌溶血素（hpmA）基因和金黄色葡萄球菌特异性pSa-442序列,运用PrimerPremier5．0分别设计3对特异性引物,预计PCR扩增的目的基因片段分别为为580bp、401bp、256bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16（4^3）正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为：94．07pg沙门菌DNA,140．85ng变形杆菌DNA,1．41ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌,4h培养后样品的最低检测限度分别为：沙门菌10^0菌落形成单位（CFU）／mL、变形杆菌10^1CFU／mL、金黄色葡萄球菌10^0CFU／mL。结论该方法特异性和灵敏度高,检验周期短,可用于对食品中多种致病菌的快速诊检和监控。