弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain Reaction

Stripe rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst . Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst . In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants.     

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked immunosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection.     

A simplified PCR-based method for the detection of Renibacterium salmoninarum utilizing preparations of rainbow trout (Oncorhynchus mykiss, Walbaum) lymphocytes.

A method for the detection of Renibacterium salmoninarum by PCR is described. A rapid, reliable procedure was developed for the extraction of DNA, which could be applied to infected kidney homogenates and head kidney lymphocyte preparations. The target for DNA amplification was a 376-bp region of the gene encoding the 57-kDa major surface antigen (MSA). The PCR was specific for R. salmoninarum and allowed the detection of 10 to 100 cells of the pathogen. Use of the PCR for the examination of experimentally infected rainbow trout showed it to be as reliable as plate culture methods for the detection of R. salmoninarum in infected kidneys.     

Comparison of three different PCR methods for detection of Brucella spp in human blood samples

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.     

嗜肺军团菌实时荧光定量PCR快速检测方法的建立

目的：建立针对嗜肺军团菌Mip基因的实时荧光定量TaqMan PCR检测方法,并进行自来水和空调冷却水模拟标本的检测评价。方法：根据嗜肺军团菌Mip基因的特异性序列设计引物和TaqMan探针,建立嗜肺军团菌的实时荧光定量TaqMan PCR快速检测方法,对方法进行灵敏度及特异性评价,并对自来水和空调冷却水模拟标本中的嗜肺军团菌进行检测。结果：建立的方法对嗜肺军团菌的检测具有高度特异性,与3种非嗜肺军团菌和6种其他呼吸道病原均没有交叉反应;基因组DNA的检测灵敏度为1.6pg／μL,模拟自来水和空调冷却水标本的检测灵敏度为10CFU／mL。结论：建立的TaqMan荧光定量PCR方法特异、灵敏、快速,适于嗜肺军团菌的日常监测和暴发疫情的应急诊断。     

用RT-PCR法快速检测鲤春病毒血症病毒基因

用逆转录多聚酶链式反应（RT-PCR）方法快速,灵敏、特异地检测鲤春病毒血症病毒（SVCV）,根据SVC病毒糖蛋白基因序列设计的引物经过RT-PCR和半嵌套PCR（semi-nested-PCR）引扩增出SVC病毒核酸中的714bp和606bp片段,与其他弹状病毒IHNV、VHSV、PFRV没有交叉,没有特异性,灵敏度检测,表明不小于1000个病毒就可检测出阳性结果。本文还对复性温度、引物、Mg^2 、Tag酶以及反转录酶的浓度对检测结果的影响进行了探讨。     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

AIM: Vibrio alginolyticus is an economically important micro-organism. The main aim of the present study was to develop a real-time polymerase chain reaction (PCR) assay for rapid, sensitive and effective quantification of V. alginolyticus in seawater and seafood. METHODS AND RESULTS: Purified DNA of V. alginolyticus, artificially inoculated seawater and seafood tissue homogenates were subjected to the gyrB-targeted real-time PCR assay. Natural seawater and seafood samples were analysed by this real-time PCR protocol. Specificity tests showed that positive result was obtained only with V. alginolyticus strains. The detection sensitivity was determined to be 0.4 pg of genomic DNA equivalent to 72 cells per PCR in pure culture and 100 cells in 1 ml of seawater or seafood tissue homogenates. Single cell detection is achieved after 3 h of sample enrichment. CONCLUSIONS: A sensitive and specific SYBR Green I-based real-time PCR assay targeting gyrB gene was successfully developed to quantify V. alginolyticus within 6 h in seawater and seafood samples. SIGNIFICANCE AND IMPACT OF THE STUDY: No report on the molecular-based method was available for quantitative detection of V. alginolyticus. This work will provide a novel method for evaluation of the risk of V. alginolyticus to marine environmental health and seafood safety.     

环介导间接PCR检测方法的建立

建立环介导间接PCR检测体系,为分子诊断提供一种新的检测工具。以质粒pUC18的核苷酸序列为模板,设计两条特异性探针,采用常规PCR技术将特异性探针标记于大豆Lectin基因的左右两端充当报告基因,此标记的报告基因与待检的pUC18质粒经杂交和缺口补平后形成一环状DNA分子,然后采用反向PCR技术扩增报告基因,建立针对pUC18质粒的环介导间接PCR检测方法。结果表明,该检测方法的检测底限为0.32 pg/μL,与常规PCR相当,并且与其他质粒和动物DNA检测无交叉反应,是一种简单、快速、灵敏、特异的PCR检测方法。     

Identification of Aeromonas hydrophila hybridization group 1 by PCR assays.

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Ovine-specific Y-chromosome RAPD–SCAR marker for embryo sexing

An accurate, sensitive, and quick (approximately 3 h) method for determining the sex of ovine embryos was developed using polymerase chain reaction (PCR) primers derived from an ovine-specific Y-chromosome random amplified polymorphic DNA marker ( UcdO43 ). The accuracy and sensitivity of the assay were first tested using genomic DNA from 10 males and 10 females of five different sheep breeds, and then tested using serial dilutions of male-in-female DNA. The assay was 100% accurate in confirming the sex of the individuals and the ovine male-specific fragment was detected in dilutions containing as little as 10 pg of male DNA in 50 ng of female DNA. The assay was also confirmed to be specific for the ovine Y-chromosome as bovine, caprine, porcine, murine, and human DNA did not amplify. The ovine embryo sexing method is a duplex PCR system that also includes ZFY/ZFX primers. ZFY/ZFX provide an internal positive control for amplification as well as a means to confirm the results obtained with the UcdO43 primers. All embryo sexing results (36/36) from our method were in agreement with the ZFY/ZFX assay results. However, while our method requires an internal control to detect PCR failure, it has the advantages of not requiring nested PCR or restriction endonuclease digestion of the PCR product, and concerns about cross-species contamination are eliminated.     

流感病毒亚单位疫苗中间体中沙门菌快速检测方法的建立

目的：建立一种能够快速、准确地检测流感病毒亚单位疫苗中间体样品中沙门菌污染的方法。方法：首先利用选择性增菌培养基对样品进行增菌培养,然后提取样品中的细菌基因组DNA,通过沙门茵特异性引物对基因组DNA进行PCR扩增,以琼脂糖凝胶电泳实现对目的片段的检测。结果：该PCR反应体系的扩增检测灵敏度可达1pg的沙门菌DNA,利用选择性增菌培养配合该PCR体系可在最快24h内实现对沙门菌的准确检测,测定结果与传统方法相符。结论：此方法应用于流感病毒亚单位疫苗中间体的沙门菌检查,较之传统的培养法结合生化鉴定的方法,大大缩短了检测周期,降低了结果判读的难度,在实际生产中有很好的应用前景。     

Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10³ to 10⁴ oocysts, and the nested PCR method was able to detect 10⁰ to 10² oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.     

应用xMAP液态芯片多重快速检测四种病原微生物的研究

目的：建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法：根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果：xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论：xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

应用xMAP液念芯片多重快速检测四种病原微生物的研究

目的:建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法:根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果:xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论:xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.