MPN-PCR法定量检测武汉鸡肉制品中的空肠弯曲菌

空肠弯曲菌（Campylobacter jejuni）是引起人类细菌性腹泻的主要人畜共患病原菌之一,严重时可导致人类格林-巴利综合征。其中鸡肉污染导致的食源性传播是人类感染的重要原因。为了调查武汉市鸡肉制品的空肠弯曲菌污染情况,运用最可能数法（MPN）及聚合酶链式反应（PCR）技术结合,即MPNPCR方法 ,对市场采集的128份鸡肉样品进行了空肠弯曲菌的定量检测,结果阳性检出率为18.75%,其中鲜鸡肉检出率为28.26%,冻鸡肉检出率为13.41%,含菌量范围为3.6-92.0 MPN/g。

油田硫酸盐还原菌APS-MPN-PCR快速定量检测方法

为了解决现有油田污水中硫酸盐还原菌检测周期长、检测费用较高的问题，应用聚合酶链式反应（PCR）技术与倍比稀释法（MPN）相结合的APS-MPN-PCR法，对硫酸盐还原菌进行快速定量检测．从污水中制备了直接用于PCR扩增的菌液，保证了定量检测的准确性；建立以腺苷酰硫酸还原酶基因（APS）为靶位点的通用探针APS7F和APS8R的反应体系和扩增条件．研究结果表明，与液体稀释培养法相比，该方法检测灵敏度提高了两个数量级，操作时间缩短（整个检测过程只需要3～4h），检测费用也降低．该方法检测结果非常稳定，真实的表征了污水中实际的SRB菌数量，可以在生产中应用．

湖泊中军团菌的巢式PCR-MPN检测法

为了直接检测环境水体中军团菌的存在,文章将巢式PCR与倍比稀释法相结合(巢式PCR-MPN法),采用两对引物扩增环境样品基因组DNA。将检测到含有军团菌的样品DNA模板进行十倍梯度稀释,由最低检测的稀释度,计算出样品中军团菌的细胞数,同时确定方法检测的灵敏度。并用克隆建库、构建系统树的方法来验证该文研究方法的特异性。研究结果表明,巢式PCR第二步后获得的序列均为军团菌序列,该方法军团菌DNA的检出限可以低至2.74 fg/μL。该研究建立的水体军团菌检测方法具有简便快捷、特异性好、灵敏度高的特点。

Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Real-time RT-PCR Assay for the Detection of Culex flavivirus

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

First TaqMan Assay to Identify and Quantify the Cylindrospermopsin-Producing Cyanobacterium <i>Aphanizomenon ovalisporum</i>in Water

Cylindrospermopsin (CYN) is an alkaloid that causes hepatotoxicity, neurotoxicity and general cytotoxicity in vertebrates. It is currently gaining widespread attention after its reported appearance in water bodies around the world. A. ovalisporum is capable of CYN-production and can form toxic blooms when favorable environmental conditions are available. We have developed for the first time a two-step qPCR assay using Taqman probes to detect and quantify potential CYN-producing A. ovalisporum in water samples. The assay was sensitive enough to discriminate between CYN-producing and non-CYN-producing A. ovalisporum in a mixed background, and discriminate between A. ovalisporum and other nostocales as C. raciborskii and A. bergii. The detection limit of the assay falls in the log linear range of 102 and 105 gene copies per reaction and is thus within the sensitivity range of previously published assays for the detection of other toxic cyanobacteria species. Our assay allows for the first time to quickly assess water quality for the presence of potentially CYN-producing A. ovalisporum and can be easily used for the purposes of monitoring water bodies.

Leishmania tropica: The comparison of two frequently-used methods of parasite load assay in vaccinated mice

Objective:To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods:BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stressinducible protein-1 with/without adjuvant.After three vaccinations,mice were challenged by Leishmania tropica promastigotes.Two months after challenge,the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods.Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium.For real-time PCR,DNA of the lymph nodes was extracted,equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers.The data of the two methods were compared by appropriate statistical methods.Results:Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice.In addition,wherever parasite load of a group was estimated high(or low)by one method,the estimated parasite load by another method was the same,although statistically significant differences were found between some groups.Conclusions:Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups.However,due to the lower errors and faster process,the real-time PCR method is preferred.

金黄色葡萄球菌荧光定量PCR法与国标培养法的比较

目的比较荧光定量PCR法、Baird-Parker平板计数法、MPN计数法,探索荧光定量PCR快速筛检金黄色葡萄球菌方法的灵敏性和可行性,为缩短食品中金黄色葡萄球菌的检测时效提供参考。方法以金黄色葡萄球菌定量检测能力验证样本、年糕、调理肉制品、酱卤肉、方便米饭为试验样品,针对三种检测方法设计灵敏性试验和人工污染试验。结果灵敏性试验三种方法在浓度105CFU/ml均有阳性检出,MPN计数法灵敏性略好,人工污染试验符合预期。结论荧光定量PCR法可为食品中金黄色葡萄球菌应急检测提供依据。

Complete genome sequence of a Rodent Torque teno virus in Hainan Island, China and establishment of a real-time PCR for detecting Rodent Torque teno virus 3

Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCR detection assay for RoTTV3.Methods: Based on the high-throughput genome sequencing analysis, specific primers were designed and the whole genome sequence was amplified by PCR and Sanger sequencing. Specific detection primers were designed based on the conserved sequences of RoTTV3. The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control. The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.Results: The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype. In this experiment, the real-time PCR detection method of RoTTV3 was established. The standard curve generated had a wide dynamic range from 1×(102-108) copies/μL, with a linear correlation (R2=1.000). The melting curve analysis using SYBR Green showed only one specific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction.Discussion: This study was the first report of the RoTTV in Hainan Islands, and its phylogenetic analysis was of great significance to the origin and evolution of RoTTV. The RoTTV3 real-time PCR detection method established in this experiment has a high sensitivity and good specificity, which lays a technical foundation for the epidemiological investigation of RoTTV3.

Feasibility of the Routine Clinical Use of a Multiplex Virus Polymerase Chain Reaction Assay Based on Blood Virus Detection in Hematopoietic Stem Cell-Transplanted Patients

Background: Multiplex virus assays are useful in immunocompromised hosts but still challenging in routine clinical settings in terms of their sensitivity, specificity, reproducibility, and time and cost performances. In recent years, we developed a qualitative multiplex virus PCR assay capable of the simultaneous detection of 13 virus species within 3 h. However, because of the multiple and concomitant nature of this virus assay, it should be validated for qualitative reliability. Materials and Methods: As a preclinical examination, this multiplex PCR was able to detect 1.25 × 103 copies/mL of 13 synthesized virus genomes and preserved same virus DNAs by the serial dilution method. Blood samples from 40 patients who underwent hematopoietic stem cell transplantation were then examined by multiplex PCR for 13 virus species, followed by quantitative real-time PCR for all 13 virus species as reference PCR when these patients developed symptoms suggestive of viral infection. Results: In 421 cumulative qualitative-quantitative tests, the multiplex PCR certainly detected 1.0 × 103 copies/mL of 5 viruses (CMV, JCV, BKV, HHV-6, ADV) that were frequently detected and thus reasonably analyzed. The positive and negative predictive values of multiplex PCR were 84.2% - 93.3% and 90.7% - 99.0%, respectively, and sensitivity and specificity were 59.0% - 83.3% and 97.2% - 99.2%, respectively, for these 5 viruses. Conclusion: From these performances, the multiplex PCR assay may be acceptable in a routine clinical laboratory setting.

国内外食品微生物快速检测技术应用进展

本文对目前国际上公认且具有良好发展空间的食品微生物检测技术和特点进行综述。包括:多聚酶链式反应PCR、实时定量PCR(real-timePCR)技术、酶联免疫法ELISA、PCR-ELISA技术、直接表面荧光滤膜计数技术、生物感应器biosensor、最近似数测定方法MPN以及Dipstick。

四种AM真菌接种剂的田间效应及其分子检测研究

采用灭菌土壤生产了 4种AM真菌接种剂。在盆栽条件下测试了接种剂的质量 ,结果显示 ,4种接种剂促进玉米生长效果明显 ,地上部分生物量均显著高于对照 (p <0 .0 1 ) ;以MPN试验检测了接种剂的侵染能力 ,结果表明每克接种剂中真菌的繁殖体数在 95～ 1 4 0 0之间。将AM真菌的预接种技术和农业生产上的营养钵育苗技术相结合 ,进行了玉米的田间试验 ,结果显示 ,玉米根系的AM真菌感染率早期增长较快 ,然后趋于平稳 ;AM真菌接种剂A(Glomusconstrictum)、C (Glomus三种菌混合 )和D (G .intraradices)对玉米籽粒产量有显著的增产效果 (p <0 .0 5 ) ;玉米籽粒的淀粉含量和磷含量也高于对照。运用特异性分子探针和nest ed PCR技术 ,从田间接种AM真菌Glomusintraradices和G .mosseae的玉米根样中粗提DNA进行特异性扩增 ,成功地从感染根段中检测到特定的接种AM真菌。本工作从分子水平为评价高效AM真菌的应用潜力、研究AM真菌之间及其与其他微生物之间的相互关系奠定了基础。

生物硝化池污水中硝化细菌的快速定量研究

实验采用聚合酶链式反应 (PCR)技术与最大几率数法 (MPN)相结合的MPN PCR法对生物硝化池污水中的硝化细菌进行快速定量 .所用的一对PCR引物是在对硝化细菌的 16SrRNA基因进行系统比较的基础上设计合成的 ,可以扩增出大小为388bp的DNA片段 .以从生物硝化池污水中抽提的含硝化细菌DNA的混合DNA为模板 ,进行PCR扩增并确定合适的扩增条件 .运用MPN

多功能农药降解基因工程菌株m-CDS-1环境释放安全评价

【目的】为了评价多功能农药降解基因工程菌株m-CDS-1的环境释放中间试验水平的安全性。【方法】通过农药检测、平板计数、Most probable number-PCR(MPN-PCR)和Denaturing Gradient Gel Electrophoresis(DGGE)等方法在江苏大丰进行了工程菌株m-CDS-1田间降解农药效果、定殖动态和对土壤微生物群落结构影响的研究。【结果】投加1.01×107CFU/g干土的工程菌株m-CDS-1在30d时均能完全降解10.71mg/kg的甲基对硫磷和1.29mg/kg的呋喃丹。平板计数表明工程菌株m-CDS-1在土壤中快速下降;MPN-PCR检测结果显示,在4d,15d和30d时,0～10cm混合土壤中该工程菌株的数目分别为2.15±0.98×106CFU/g干土,3.70±4.66×104CFU/g干土和检测不出。工程菌株m-CDS-1的投加不会对土壤可培养三大微生物菌群数量产生显著影响;基于细菌16S rDNA V3区的DGGE分析结果表明,施加农药对细菌菌落结构有显著影响,4d,11d,30d时农药施用区与空白对照区的图谱相似性分别为17.16%,49.81%和75.01%,但到60d时的相似性为98.62%。工程菌株m-CDS-1释放在前期对细菌群落结构有一定影响,4d,11d和30d工程菌株释放区相对于空白的相似性分别为49.57%,38.30%和83.30%。在60d时,空白、施药和施菌小区的图谱相似性都在90%以上。【结论】工程菌株m-CDS-1不仅可同时高效降解甲基对硫磷和呋喃丹,仍保持了实验室内的原有特性,而且不会成为优势菌群长期在土壤环境中存在,也不会对土壤微生物群落结构造成长期影响。

油田硫酸盐还原菌快速定量检测方法

现有油田废水中硫酸盐还原菌检测周期长、检测费用较高，本研究应用聚合酶链式反应（PCR）技术与倍比稀释法（MPN）相结合的DSR-MPN-PCR法，对硫酸盐还原菌进行快速定量检测．从废水中制备了直接用于PCR扩增的菌液，保证了定量准确性；建立以硫酸盐还原菌亚硫酸盐还原酶基因（Dsr）为靶位点的通用探针DSR1F和DSR5R的反应体系和扩增条件．结果表明，该方法检测灵敏度明显比液体稀释培养法高2个数量级，真实地表征了废水中实际的SRB菌数量，整个操作过程需要3-4h。检测结果非常稳定，降低了检测费用，可以在生产中应用．

基于nir S基因的反硝化细菌快速定量检测

为解决现有油田污水以及反硝化相关的生物反应器中,反硝化细菌定量检测周期长、检测费用高的问题,以nir S基因为靶基因,采用聚合酶链式反应(PCR)技术与倍比稀释法(MPN)相结合的nir S-MPN-PCR法,对反硝化细菌进行快速定量检测研究.从污水中制备直接用于PCR扩增的菌液,保证定量准确性;以832F和1606R为通用引物,优化反应体系和扩增条件.结果表明,该方法检测结果明显比MPN-Gries法灵敏,真实地反映污水中实际的DNB菌的数量,整个操作过程需要3～4h,检测结果非常稳定,降低了检测费用,可以在生产工艺中应用.

JAK2-V617F突变初步研究

目的用等位基因聚合酶链反应(AS-PCR)方法检测Bcr-abl阴性的骨髓增殖性肿瘤(MPN)、骨髓增生异常综合征(MDS)、骨髓增生异常综合征/骨髓增殖性肿瘤(MDS/MPN)、急性髓系白血病(AML)、急性淋巴细胞白血病(ALL)、Bcr-abl阳性的慢性粒细胞白血病(CML)患者和正常健康人对照的外周血白细胞JAK2-V617F基因点突变情况,了解该突变在不同疾病中的表达,并建立敏感特异高效的JAK2-V617F突变的临床检测方法。方法采用AS-PCR方法检测基因组中JAK2-V617F突变,并经基因测序确定。所有MPN患者均行骨髓活检了解其纤维增生度。结果MPN各型中真性红细胞增多症(PV)阳性率为93.8%;原发性血小板增多症(ET)阳性率为60.0%;原发性骨髓纤维化(IMF)阳性率为0;MDS阳性率为4.5%;MDS/MPN阳性率为12.5%;AML、ALL、CML及正常对照均为阴性。MPN各型之间、MPN与其它疾病之间,阳性率差异有显著性(P<0.05)。JAK2-V617F突变阳性的PV及ET患者白细胞数较阴性患者高,差异有显著性(P<0.05)。且阳性患者骨髓纤维组织增生较阴性者明显,差异有显著性(P<0.05)。AS-PCR法检测阳性率高于直接测序法,差异有显著性(P<0.05)。结论JAK2-V617F是检测MPN尤其是PV的分子遗传学标志,同时与白细胞数、骨髓纤维化程度成正相关,有助于临床诊断及疾病预后判断。AS-PCR法是检测JAK2-V617F突变的敏感、特异的方法,可以成功应用于临床检测。

淄博市市售生食贝类中副溶血性弧菌污染水平调查

为了解山东省淄博市市售生食贝类中副溶血性弧菌的污染状况和毒力基因的携带情况,分别于2019年8、9、10月从不同区县的零售市场采集牡蛎和毛蚶样品共40份,运用副溶血性弧菌MPN定量法和多重荧光MPN-PCR法对副溶血性弧菌的污染状况和毒力基因的携带情况进行调查。结果显示,副溶血性弧菌(tlh基因)检出率为47.50%(19/40),其中污染量≥110MPN/g的样品占26.00%(5/19);毒力基因(tdh、trh)阳性15份,携带率37.50%(15/40),其中牡蛎携带率为45.00%(9/20),毛蚶携带率为30.00%(6/20)。故得出结论,淄博市市售牡蛎和毛蚶中副溶血性弧菌的污染水平较高,存在一定的食品安全风险。