猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示：该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到10^2.0 TCID₅₀/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。     

基于定向定量堆放的马铃薯收获机设计

在研究小型马铃薯收获机的基础上,设计了一款能根据收集薯块质量往特定方向堆放的马铃薯收获机,主要由挖掘装置、输送装置及定向定量堆放装置等组成。在马铃薯收获机的后部设计一个四分区临时集薯器,能根据所收获马铃薯的质量来打开集薯器,将收集的马铃薯向中间或者一侧堆放,从而实现了马铃薯的连续挖掘及收集薯块的定向定量间隔堆放,可在较大程度上降低人工捡拾的工作量,同时避免拖拉机碾压伤薯。     

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.     

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。     

猪捷申病毒荧光定量RT-PCR检测方法的建立与应用

参考Gen Bank中猪捷申病毒(PTV)基因序列,通过比对分析,设计引物和Taq Man探针,建立荧光定量RT-PCR检测方法。该方法的最低检测灵敏限度为12.6拷贝/μL,组内组间重复试验的变异系数均小于2%,具有较好的特异性,对猪常见病原如猪瘟病毒、猪传染性胃肠炎病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒和伪狂犬病病毒等均没有特异性扩增。用本研究建立的荧光定量RT-PCR方法检测来自猪场的156份猪粪便,阳性检出率为38.5%,与套式RT-PCR符合率达100%,表明本试验建立的荧光定量RT-PCR方法适用于PTV的快速批量筛检,为PTV感染的流行病学调查提供了必要的技术手段。     

金针菇异戊烯基焦磷酸异构酶基因的克隆及表达分析

PCR扩增了金针菇(Flammulina velutipes)异戊烯基焦磷酸异构酶基因(FvIDI)的DNA全长和开放阅读框序列.该基因的ORF全长753 bp,DNA全长923 bp,含有3个内含子和4个外显子,编码250个氨基酸,金针菇异戊烯基焦磷酸异构酶相对分子质量为28450,等电点为5.05.采用实时荧光定量PCR技术研究FvIDI基因在不同发育时期和不同温度下的表达量,结果显示,FvIDI基因在采收期菌盖中表达量最高,4℃诱导处理2～4 h和37℃诱导处理2～6 h的表达量明显高于常规温度21℃处理.     

甜瓜黄斑病毒SYBR Green Ⅰ实时荧光定量PCR方法

为建立检测甜瓜黄斑病毒(melon yellow spot virus, MYSV)的SYBR Green Ⅰ实时荧光定量PCR(qPCR)方法。基于MYSV核衣壳蛋白基因保守序列设计qPCR特异性引物对,针对引物退火温度、引物浓度、特异性和敏感性进行系列优化。结果显示,优化后的qPCR方法最适退火温度为61.3℃,最适引物浓度为0.65μmol·L^-1,特异性强,灵敏度高,比PCR高100倍。以携带目的基因片段的重组质粒为标准品,构建的qPCR标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.999 7。实验样品验证表明建立的qPCR方法可用于MYSV的定量检测。