布鲁氏杆菌PCR诊断方法的研究进展

布鲁氏杆菌病(Brucellosis),简称布病是由布鲁氏杆菌引起的一种人畜共患全身性传染病,是世界上最常见的细菌人畜共患病。猪、牛、羊等主要家畜以及野生动物均可感染该病。人群接触带菌动物或食用病畜肉、乳制品等也均有感染的可能。动物和人类感染布病后会对生命安全和财产造成重大的威胁和损失。目前,病原体的分离培养、血清学方法和PCR技术是布病诊断的三大主流技术。但是,由于人类布鲁氏菌病的非特异性临床特征、血培养生长速度缓慢及其血清诊断的复杂性,因此对布病做出及时准确的诊断仍是我们现阶段面临的主要挑战。本文主要就PCR技术诊断布鲁氏杆菌的研究进展做一综述,以期为后续的研究提供理论依据和参考。     

奶牛乳铁蛋白基因部分序列的PCR—SSCP分析

本研究采用PCR－SSCP方法对奶牛乳铁蛋白基因的第7、12外显子和5′调控区部分序列进行多态性分析，发现该基因5′调控区一段长227bp的DNA片段上存在多态性。经过对突变纯合（BB）的两头奶牛个体进行测序分析后证明，位于起始位点上游CAAT框－926和－915位置分别具有G→A及T→G点突变，同时在－839和－811位置分别具有C和T单碱基的。等位基因A、B的频率分别为0.898和0.102。     

基因芯片检测转基因油菜

在设计转基因油菜(Brassica napus)的基因芯片检测方法时，根据油菜中所转入的外源基因，选择了CaMV35S启动子、FMV35S启动子、Nos终止子、Bar基因、Barnase基因、Barstar基因、EPSPS基因、GOX基因、PAT基因和内源基因Fbp等设计了引物对与探针，并制备了寡核甘酸芯片，通过多重PCR对样品核酸进行扩增和荧光标记后，将PCR产物与芯片杂交，检测油菜样品中所含的外源基因。结果表明，实验有较好的特异性和重复性，在检测低含量的转基因油菜时灵敏度可达到0．5％。由于采用了多重PCR和芯片的多基因并行杂交的技术，一次可同时检测10个基因，在检测多品种混合的转基因油菜商品时具有独特优势。     

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.     

Study on Expression Difference of IGFBP-5 Gene in Different Tissues of Kazakh and Yanqi Horses

The study aimed to explore the mRNA expression pattern of insulin-like growth factor binding protein-5 (IGFBP-5) gene in different tissues of Kazakh and Yanqi horses.The expression of IGFBP-5 gene in different tissues of heart,liver,spleen,lung,kidney,small intestine,large intestine,cecum,intercostal muscles,longissimus dorsi muscles,brachialis muscle and gluteus in two horses were detected by Real-time quantitative PCR and compared the mRNA expression in the same tissues of two breeds.The results showed that the expression of IGFBP-5 in longissimus dorsi muscle and brachialis muscle of two breeds were significantly higher than other tissues including heart,liver,spleen,lung,kidney,small intestine,large intestine and cecum (P<0.05),and was the lowest in large intestine.The expression of IGFBP-5 in kidney,small intestine,large intestine,cecum,longissimus dorsi muscle and brachialis muscles of Yanqi horse were higher than in the same part of Kazakh horse,and among those in longissimus dorsi muscle and large intestine of Yanqi horse were extremely significantly higher than in the same part of Kazakh horse (P<0.01),and in small intestine and cecum of Yanqi horse were significantly higher than in the same part of Kazakh horse (P<0.05).The test was for further researching the biological function of IGFBP-5 gene,and it could provide a theoretical basis for genetic improvement of production performance of horse in our country.     

Development of A Dual Real-time PCR for the Rapid Detection of Listeria monocytogenes

To establish a rapid assay for Listeria monocytogenes(LM) detection,a Real-time PCR method was developed targeting iap gene of LM.The results showed that the test for 15 bacteria strains,only LM was positive,indicated that the method had high specificity.In addition,the sensitivity of Real-time PCR was 6.5 CFU/mL.Stability and reproducibility of the test showed that the coefficient of variation for the same sample repeat the Ct values were less than 2%.Furthermore,a total of 3 positive samples for LM were detected from 139 clinical samples by the method,which was in accordance with the testing result by GB 478930-2010 standard detection protocol.Therefore,the Real-time PCR method provides a novel rapid,sensitive and good repeatability detection method for LM infection.     

Expression Analysis of THBS3 Gene in Different Tissues and Skeletal Muscles at Different Periods from Landrace and Tongcheng Pigs

To study the expression pattern of THBS3 gene in different tissues and during skeletal muscle development, the THBS3 gene expression in different tissues and skeletal muscles during prenatal periods (33, 45, 65, 70 and 90 d) and postnatal periods (0, 9, 30, 60, 120 and 160 d) from Landrace and Tongcheng pigs were detected by Real-time quantification PCR.The results showed that THBS3 gene widely expressed in all tissues examined, exhibiting similar spatial expression patterns with expression peaks in lung in both pig breeds except in stomach and intestine.Moreover, although THBS3 gene showed a significant higher expression level in gestation than after birth in Landrace and Tongcheng pigs (P<0.05), it exhibited different expression patterns between Landrace and Tongcheng pigs, the expression peak was detected at gestation day 45 in Landrace pig, while was detected at gestation day 65 in Tongcheng pig.The results suggested that THBS3 gene involved in skeletal muscle growth and development in pigs, as well as the regulation of asynchronization of skeletal muscle development in different pig breeds.     

Analysis on Polymorphisms of Three CNV Regions in Five Pig Breeds

The paper was aimed to investigate the polymorphism of copy number variation (CNV) in different pig breeds.Three CNV regions of CNVR91,CNVR92 and CNVR143 were chosen from the porcine SNP60 chip genotyping results.The polymorphisms of three CNVs were determined by Real-time quantitative PCR method,taking five pig breeds as samples,including Yorkshire pig,Xiang pig,Kele pig,Nuogu pig and Rongchang pig breeds.The results showed that the dominant status of CNVR91 was loss in Xiang pig,while it was normal in other four pig breeds.The major type of CNVR92 was deletion in Xiang pig,Yorkshire pig,Kele pig and Rongchang pig breeds,with a high normal percent in Nuogu pig.For CNVR143,the dominant event was gain in Xiang pig and Nuogu pig breeds,but it was not diverse in other three pig breeds.These results indicated that three CNV regions emerged with polymorphism in five pig breeds,which might have effects on gene expression in CNV regions and physiological function by dosage effect especially in Xiang pig,Nuogu pig and Kele pig breeds.     

猪细环病毒检测方法研究进展

猪细环病毒（TTSuV）是近年来新发现的DNA病毒,在世界猪群中广泛存在,严重威胁着养猪业的发展,而且有潜在跨物种传播给人类的可能性,引起了养猪业的极大关注。通过综述TTSuV近年来检测方法研究进展,以期对科研工作者和广大养殖业主更好地防治该病提供参考。     

基于实时荧光定量PCR技术检测桑丁香假单胞菌

由桑丁香假单胞菌(Pseudomonas syringae pv.mori,以下简称Psm)引起的桑疫病是一种毁灭性的桑树细菌性病害,建立对病原菌的早期检测技术,有利于及时制定病害的防控技术措施。依据致病性相关基因hrp Z的片段设计的一对引物能够在Psm中特异性地扩增出195 bp大小的条带,而用于对其他致病变种的丁香假单胞菌和其他种属病原菌的扩增则没有此条带。利用Psm14-7菌株基因组DNA为模板进行实时荧光定量PCR(q PCR)扩增,模板DNA质量浓度在6×10~(-5)~6 ng/μL范围内与扩增所得Ct值呈现良好的线性关系,线性方程为y=-3.224 69x+14.034 45(R~2=0.997 09),检测的最低限为(60±0.001 2)fg/μL;以Psm14-7菌液为模板进行q PCR,菌液浓度在1×10~2~1×10~8CFU/m L范围内与Ct值呈现良好的线性关系,线性方程为y=-4.562 15x+46.911 67(R~2=0.988 72),最低检测限为(10~2±0.008 3)CFU/m L。于桑树植株人工接种Psm后不同时间,对出现各种症状植株中的菌群数量进行q PCR检测,结果显示:接种后的第4~8天可能为病原菌诱导桑树的过敏性反应和系统获得性抗性关键时期,影响到了Psm的增殖速度;能引起健康桑树暴发桑疫病的致病菌最低浓度为(1.5×10~8±0.076 8)CFU/g。建立的q PCR检测方法,对由Psm引起的桑疫病的田间早期诊断和及时防控具有一定指导意义。