单增李斯特菌检测基因芯片的研制与评价

目的 研制快速、特异、灵敏的检测单增李斯特菌的基因芯片.方法 选择gyrB、ISR、16S rRNA、23S rRNA、hlyA、iap和prfA作为单增李斯特菌的检测靶基因,研制一种Oligo探针基因芯片,对18个不同种属来源的已知参考生物样品进行检测和鉴定,并且采用对比试验、重复性试验、灵敏度试验和特异性试验对该芯片进行验证评估.结果 通过对比发现IDT合成的70 mer Oligo芯片探针在芯片打印与芯片检测两个方面较优.比较10、40和80 μmol/L 3个Oligo探针点样浓度,结果 显示10 μmol/L的探针点样浓度已能获得很好的芯片检测结果.单增李斯特菌检测芯片具有较好的重复性;样品检测绝对量下限为0.9 ng DNA左右.结论 Oligo基因芯片可以快速准确地检测单增李斯特菌.     

实时荧光RT-PCR检测活性单核细胞增生李斯特菌方法的建立

目的 采用逆转录结合实时荧光定量PCR技术,建立一种快速、准确、特异甄别单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李氏菌)死活状态的定量方法.方法 根据单增李氏菌hlyA基因序列设计引物和探针;对实时荧光PCR反应体系进行优化后,提取菌体mRNA,通过随机引物进行逆转录反应;产生的cDNA通过实时荧光定量PCR进行鉴定.进一步评价逆转录结合实时荧光定量PCR方法的特异性、灵敏度、重复性后,对20份模拟双盲样本进行检测.结果 本实验所建立的逆转录结合实时荧光定量PCR方法可准确、特异地检测单增李氏菌,其他菌株和失活的单增李氏菌均无阳性结果出现;该方法检测纯菌和模拟样本的灵敏度分别可达到10 CFU/ml和1000CFU/ml;定量检测的批间和批内的变异系数均小于5%;对20份模拟样本进行检测,其中10份含有活性单增李氏菌样本的检测结果均为阳性,其他含有失活单增李氏菌的5份样本和其他致病菌的5份样本检测结果为阴性.结论 本文建立的检测活性单增李氏菌实时荧光定量PCR方法快速、准确,结果可靠,实用性强,可进行定量分析,为食品安全监测和现场流行病学调查提供较好的分析手段和完整的数据.     

单核细胞增生性李斯特菌iap基因克隆和PCR条件优化

目的 构建单核细胞增生性李斯特菌Listeria monocytogenes 54002-4株p60蛋白iap基因原核克隆载体.方法 利用自行设计的引物通过梯度PCR优化扩增条件,扩增出单核细胞增生性李斯特菌54002-4株的iap基因.在iap基因的5'端和3'端分别引入BamH Ⅰ和Xho Ⅰ 2个酶切位点,琼脂糖凝胶电泳分析、回收PCR产物,并将回收纯化的PCR产物与pMD 18-T载体进行连接.将该重组质粒转化人大肠杆菌JM109感受态细胞,经1 mmol/L IPTG诱导4～6 h后,观察转化效果.结果 培养无色菌落细菌,提取质粒,经PCR鉴定和核苷酸序列测定后确定获得阳性重组质粒pMD18-T-Iap.结论 通过梯度PCR摸索出最佳反应条件,建立PCR优化条件和方法,经转化获得iap基因克隆载体.     

LAMP和PCR法检测痢疾志贺菌的特异性和灵敏性比较

目的建立环介导等温扩增技术（LAMP）快速检测痢疾志贺菌,并对其特异性、灵敏度与传统PCR进行比较。方法以痢疾志贺菌侵袭性质粒抗原H基因（ipaH）为靶序列,设计6条特异性引物（内引物、外引物和环引物各2条）,优化LAMP反应体系及反应条件,检测LAMP反应的灵敏度、特异性及模拟样品,同步与PCR进行比较。结果 LAMP法和PCR均能特异性地扩增出志贺菌靶DNA,而其他非志贺菌均未扩增出特有条带。检测细菌纯培养物和模拟食品的灵敏度LAMP法分别为5.3×101cfu/ml、6.8×101cfu/ml,PCR法分别为5.3×102cfu/ml和6.8×102cfu/ml。结论利用LAMP技术,可快速、灵敏、简便地检测痢疾志贺菌,与PCR方法比较,特异性强、操作简便、检测成本低、耗时短,有望发展成为快速检测痢疾志贺菌的有效手段。     

α变形菌纲磁螺菌属TaqMan探针实时荧光定量PCR快速检测方法的建立

目的建立一种快速有效检测环境中q变形菌纲磁螺菌属细菌的方法。方法本研究以α变形菌纲磁螺菌属保守且特异的16SrRNA片段为靶序列,化学合成基因序列,制备重组质粒作为磁螺菌属检测的标准品;用实时荧光定量聚合酶链反应（FQ—PCR）技术,针对目的片段基因设计特异性引物和TaqMan荧光探针,进行实时荧光定量PCR检测,运用统计学方法评价该方法的特异性、敏感性及重复性。结果本实验成功构建了质粒PUC19-QC,引物、探针特异性良好,标准曲线在5．10×10^1～5．10×10^7拷贝数之间具有较好的线性关系,相关系数R2=0．999。该法最低可检测到5．10x10个DNA拷贝数,灵敏度明显高于普通PCR;重复性试验表明．荧光定量PCR检测结果Ct值波动范围较小,α值的标准差均小于0．23,表明该法重复性好,可靠性高。析因方差分析表明不同稀释度之间的ct值差异有统计学意义（F=3125．305,P〈0．001）,三个批次的再现性良好,差异无统计学意义（F=0．057,P=0．945）,而浓度分组与时间之间也无交叉效应（F=0．533,P=0．873）。结论本实验所建立的TaqMan探针实时荧光定量PCR检测技术,能够快速有效地检测仅变形菌纲磁螺菌属细菌,且检测结果稳定,受外界条件如时间、气温等影响小。     

不明原因肺炎监测中SARS-CoV实时荧光RT-PCR检测方法的建立

目的 建立SARS-CoV的TaqMan探针实时荧光RT-PCR检测方法并进行评估,为不明原因肺炎监测中SARS-CoV感染的排查提供实验室检测依据.方法利用SARS-CoV的1b基因和NP基因的特异性序列设计引物和探针,两条探针5'端标记FAM,3'端标记TAMRA.与国家流感中心发布的高致病性人禽流感H5N1的实时...     

BCR/ABL重组质粒实时荧光定量PCR标准品的制备

目的：用T-A克隆法构建含BCR/ABL融合基因的重组质粒,并用实时定量PCR（RQ-PCR）方法制备标准品。方法：通过培养细胞,提取总RNA并逆转录为cDNA后做PCR,电泳胶回收纯化,T-A克隆与pUCm-T载体连接,转染DH5a菌,蓝白斑筛选阳性菌落后,大量提取质粒,再进行RQ-PCR,最后制得BCR/ABL的重组质粒标准品。结果：蓝白斑筛选实验、PCR扩增均证实BCR/ABL融合基因重组到pUCm-T载体上,经RQ-PCR定量后得到BCR/ABL重组质粒标准品的标准曲线。结论：该方法能大量制备质粒标准品,并且可被推广应用。     

海藻希瓦菌中发现一种新的qnrA7基因亚型

目的 研究海藻希瓦菌中质粒介导的喹诺酮类耐药基因的分布和特性.方法 PCR检测qnr、qepA、aac(6')Ib-cr基因,阳性产物进行DNA测序以确定其基因型,接合转移试验探讨质粒介导的喹诺酮类耐药基因的体外转移性,E-test法测定菌株MIC,提取质粒对qnrA基因进行初步定位.结果 海藻希瓦菌中检出qnrA基因,为新发现的亚型,命名为qnrA7,GenBank登录号为GQ463707,未检出qnrB、qnrS、qnrC、qnrD、qepA、aac(6')-Ib-cr基因;qnrA7位于约33 kb的质粒上,但体外接合转移试验未成功;该菌对喹诺酮类药物敏感.结论 海藻希瓦菌质粒上检出新的qnrA基因亚型,其作为qnr基因的环境宿主值得关注.     

基于载体减毒单增李斯特菌的弧菌疫苗菌株构建

目的：利用减毒单增李斯特菌作为疫苗载体的优势,拟构建基于载体减毒单增李斯特菌的弧菌疫苗菌株。方法：使用单增李斯特菌表达载体pERL3过表达弧菌中共同的靶标抗原Ompk;利用PCR和RT-PCR技术分别对过表达菌株鉴定和抗原基因的检测。结果：PCR结果显示成功构建3株Ompk过表达菌株Lm-Ompk（L-O）、Lm-Lmo0576-Ompk（L-L-O）和Lm-P-Ompk（L-P-O）;测序结果显示过表达菌株中外源基因的序列与NCBI 结果相似性为100%;RT-PCR结果显示Ompk基因在3株过表达菌株中的转录水平均极为显著（P<0.001）;在抗生素条件下,Ompk基因在各过表达菌株中的表达水平显著高于在非抗性条件下的转录水平（P<0.05）。结论：成功构建了3株基于载体减毒单增李斯特菌的弧菌疫苗菌株,弧菌中共同的靶标抗原Ompk在3株重组菌株中均可以稳定表达。     

发热伴血小板减少综合征布尼亚病毒实时荧光RT-PCR检测方法的建立

目的 建立新型发热伴血小板减少综合征布尼亚病毒的TaqMan探针实时荧光RTPCR检测方法并进行评估,为发热伴血小板减少综合征监测中新型布尼亚病毒感染的排查提供实验室检测依据.方法 利用新型布尼亚病毒S片段基因的特异性序列设计引物和探针,探针5'端标记FAM,3'端标记TAMRA,优化反应体系与反应条件,并对不同浓度含...     

Detection of Listeria monocytogenes in food using a combined enrichment/real-time PCR method targeting the prfA gene

A combined enrichment/real-time PCR method for the detection of Listeria monocytogenes is presented. The method is based on a conventional PCR assay targeting the prfA gene, which has been validated and suggested as an international standard PCR method for identifying L. monocytogenes in food. This real-time PCR assay includes an internal amplification control. Inclusivity and exclusivity were 100% each when testing 100 L. monocytogenes isolates, 30 Listeria spp. isolates other than L. monocytogenes, and 29 non-Listeria isolates. The theoretical detection limit was one copy of the target gene per PCR reaction and the practical detection limit was about 5 copies per PCR. Using the combined enrichment/real-time PCR method, 7.5 CFU/25 ml of artificially contaminated raw milk, and 9, 1, and 1 CFU/15 g of artificially contaminated salmon, paté, and green-veined cheese, respectively, were detected. When analyzing 76 naturally contaminated food samples of various types and comparing the results with the ISO 11290-1 standard method, the relative accuracy was 96%, the relative specificity 100%, and the relative sensitivity, 76.9%.     

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.     

Identification of Listeria species by microarray-based assay

We have developed a rapid microarray-based assay for the reliable detection and discrimination of six species of the Listeria genus: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi. The approach used in this study involves one-tube multiplex PCR amplification of six target bacterial virulence factor genes (iap, hly, inlB, plcA, plcB, and clpE), synthesis of fluorescently labeled single-stranded DNA, and hybridization to the multiple individual oligonucleotide probes specific for each Listeria species and immobilized on a glass surface. Results of the microarray analysis of 53 reference and clinical isolates of Listeria spp. demonstrated that this method allowed unambiguous identification of all six Listeria species based on sequence differences in the iap gene. Another virulence factor gene, hly, was used for detection and genotyping all L. monocytogenes, all L. ivanovii, and 8 of 11 L. seeligeri isolates. Other members of the genus Listeria and three L. seeligeri isolates did not contain the hly gene. There was complete agreement between the results of genotyping based on the hly and iap gene sequences. All L. monocytogenes isolates were found to be positive for the inlB, plcA, plcB, and clpE virulence genes specific only to this species. Our data on Listeria species analysis demonstrated that this microarray technique is a simple, rapid, and robust genotyping method that is also a potentially valuable tool for identification and characterization of bacterial pathogens in general.     

Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene

Listeria monocytogenes is a bacterial pathogen that can invade the central nervous system (CNS), causing meningoencephalitis and brain abscesses. The diagnosis of CNS listeriosis, based on the isolation of the bacteria in the cerebrospinal fluid (CSF), can be difficult because of previous antibiotic treatment and a low number of bacteria in the CSF. To improve the sensitivity of microbiological diagnosis, we have developed a real-time PCR assay for detecting and quantifying L. monocytogenes DNA in the CSF. The designed primers specifically amplify the L. monocytogenes hly gene, which encodes listeriolysin O, a pore-forming cytolysin. The PCR assay for the hly gene (PCR-hly) provides reproducible quantitative results over a wide dynamic range of concentrations and was highly sensitive while detecting a single gene copy/ml. By assaying a large panel of bacterial species, including species secreting pore-forming cytolysin, we determined the specificity of the PCR-hly, which exclusively detects the L. monocytogenes DNA. We then analyzed 214 CSF samples from patients suspected of having CNS listeriosis. PCR-hly was positive in all cases in which L. monocytogenes was isolated by culture. Positive PCR-hly of the CSF was also obtained for five additional, clinically confirmed cases of CNS listeriosis for which bacterial cultures were negative presumably due to previous treatment with antibiotics. As a complement to classical bacteriological CSF culture, our designed real-time PCR-hly assay proved to be valuable by enhancing the rapidity and the accuracy of the diagnosis of CNS infection by L. monocytogenes. In addition, the quantitative results provided may, in some instances, be useful for the follow-up of patients under treatment.     

Development of polymerase chain reaction assays for detection of Listeria monocytogenes in clinical cerebrospinal fluid samples.

In order to improve the diagnosis of Listeria meningitis or meningoencephalitis, especially in patients who have received antibiotics before their cerebrospinal fluid (CSF) has been examined, two assays for the detection of Listeria monocytogenes based on the polymerase chain reaction (PCR) were evaluated. After a standard PCR, the amplified DNA was detected either by a second round of PCR with internal primers followed by gel electrophoresis and ethidium bromide staining (nested PCR) or by dot blot hybridization to an internal digoxigenin-labeled probe (PCR-dot blot). For PCR, two sets of primers within the invasion-associated protein gene (iap gene) were chosen. They allowed for the highly specific detection of all L. monocytogenes reference strains tested (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, and 7). These primers did not detect amplification products from various other gram-positive or gram-negative bacterial DNAs or human DNA. The sensitivities of both assays were assessed on sterile CSF samples that were artificially seeded with serial dilutions of L. monocytogenes serotype 4b cells. By both methods the limit of detection was less than 10 cells in the initial reaction. Since the nested PCR is more prone to contamination because of manipulation of the amplified products, a standard PCR assay followed by dot blot hybridization was applied to 52 CSF samples in a retrospective study. Of 28 CSF samples which were sterile or positive for bacteria other than Listeria species, 24 were PCR negative. In contrast, from 17 patients with culture-proven Listeria meningitis, 14 of 17 initial CSF samples were PCR positive, as were 3 of 7 culture-negative followup CSF samples taken after patients received antibiotics. These results support the usefulness of this approach in the diagnosis of Listeria meningitis, in particular, when antibiotic administration precedes culture of CSF.     

Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam

Shigella spp. are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods. To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA. Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by population-based surveillance of patients seeking care for diarrhea were processed by real-time PCR. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli. Shigella spp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001). The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001). The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections. The clinical presentation of shigellosis may be directly related to the bacterial load.     

Quantification of Staphylococcus aureus in unpasteurised bovine and caprine milk by real-time PCR

A sensitive and reproducible real-time PCR assay targeting the nuc gene of Staphylococcus aureus was applied for quantification of this microorganism in artificially and naturally contaminated raw milk samples. The S. aureus cell equivalents (SCEs) estimated by the real-time PCR method were two log scales higher than colony forming units (CFUs) estimated from a plate count method in artificially contaminated milk. The repeatability of the real-time PCR assay including the DNA isolation procedure was assessed by analysing the data derived from naturally contaminated samples. The relative standard deviation of the log-transformed data of four real-time PCR measurements including duplicate DNA isolations ranged between 11.3 and 1.0%. When analysing 80 bovine and 107 caprine naturally contaminated raw milk samples, the real-time PCR method yielded 19.3% more positive samples than the plate count method. With the exception of one sample, SCEs were always higher than CFUs. The difference between SCEs and CFUs was highly variable, and it was not possible to correlate real-time PCR-derived SCEs and CFUs. However, as each SCE detected by real-time PCR indicates a S. aureus cell, which is or has been present in the sample, this method offers the advantage of a retrospective analysis even of processed samples to aid food poisoning-related risk assessment.     

Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method

Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA; no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1- and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1- and HSV-2-specific LAMP methods to be 1,000 and 10,000 copies/tube, respectively. After initial validation studies, 18 swab samples (in sterilized water) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined. HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 samples that also demonstrated viral DNA detection by real-time PCR. Nine of these 10 samples exhibited HSV-1 LAMP products by turbidity assay. Furthermore, both the agarose gel electrophoresis and the turbidity assay directly detected HSV-1 LAMP products in 9 of the 10 swab samples collected in sterilized water. Next, we examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions. HSV-2 was isolated from all of the samples and visualized by either agarose gel electrophoresis or turbidity assay.     

Method To Detect Only Live Bacteria during PCR Amplification

Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 10(8) heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 10(8) heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 10(3) to 10(7) antibiotic-treated L. monocytogenes cells but could detect 10(2) live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 10(3) to 10(7) heat-treated cells but could detect 10(1) live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.