家蚕微孢子虫诊断新技术的研究

为了正确诊断家蚕微孢子虫(Nosema bobycis, N.b),共征集了11种微孢子虫作为诊断的材料。根据N.b.孢子(日本株)的DNA序列,设计一对引物,以N.b.DNA为模板进行PCR扩增得到一个约317bp的片段,将此片段克隆到大肠杆菌DH5 α中,提取重组质粒、酶切、回收目的片段,经地高辛标记为探针,对N.b.等11种微孢子虫DNA进行核酸杂交检测,只有N.b.DNA呈阳性反应。检测灵敏度均达到1ng DNA水平。     

枇杷iPBS分子标记的PCR体系优化与引物筛选

枇杷的DNA为模板进行iPBS-PCR扩增，采用预实验中效果较好的iPBS分子标记引物2241，对枇杷iPBS-PCR反应体系中的dNTP、Mg2+、引物、模板用量进行优化，根据检测结果确定枇杷iPBS-PCR使用：10×Taq buffer 2μL，25mM Mg2+ 1.6μL，2.5mM dNTP 1.6μL，10μmol/L Primer 1μL，5U/μL Taq酶0.2μL，模板DNA 5ng 的20μL反应体系。反应程序中的退火温度可以参考iPBS引物理论Tm值。对33条iPBS引物扩增测试的结果显示在该反应体系下可以筛选到22谱带清晰、多态性好的引物，可用于枇杷的分子标记分析。     

家蚕微孢子虫PCR检测的研究

根据家蚕微孢子虫（Nosemebombycis,N．b）孢子的DNA序列，设计合成一对引物，对家蚕微孢子虫孢子及其近缘种孢子的DNA进行PCR检测。结果表明只有家蚕微孢子虫孢子DNA获得特异性扩增区带，大小为317bp，可以区别于Nosemasp.MG1和MG2、柞蚕微孢子虫、Vairimorphanecatrix及Pleistophoraanguillarum等。对N.b孢子DNA检测灵敏度达1ng水平。     

百脉根ISSR-PCR反应体系的建立与优化

运用L16(45)正交设计对百脉根ISSR反应的5个因素,即DNA模板浓度、dNTPs浓度、引物浓度、Mg2+浓度、TaqDNA聚合酶浓度在4个水平上进行优化实验,通过不同反应体系扩增效果比较,最终确定在20uL体系中各反应物的最适含量为:40ng模板DNA、2μL 10×PCR Buffer、0.15mmol/LdNTPs、0.75μmol/L ISSR引物、1.5mmol/L MgCl2、1UTaqDNA聚合酶。该体系的建立为今后利用ISSR技术进行百脉根属种质资源的遗传多样性分析奠定了技术基础。     

检测柑橘黄龙病的四对常规PCR引物特异性和灵敏度比较

柑橘黄龙病是目前危害柑橘产业的重要危险性病害，实验室中通过PCR技术检测柑橘黄龙病是最有效的方法，因此，确定一组特异性好、灵敏度高的引物至关重要。本文通过荧光定量PCR标定柑橘黄龙病菌核酸浓度的方法，对目前各实验室常规PCR应用比较广泛的4对引物（P400F/R、P535F/R、OI1/OI2，16SF/R）做了特异性和灵敏度的比较。结果发现4对引物均能排除其它来源核酸的干扰，表现出明显的特异性，但在灵敏度方面4对引物差别很大，灵敏度结果是依次为：16SF/R＞P400F/R ＞P535F/R=OI1/OI2，灵敏度最高的16SF/R引物检测柑橘黄龙病菌核酸含量约为1.87×10﹣2 ng/μl。本研究结果为实验室开展检测柑橘黄龙病研究提供了很好的理论与实践基础     

RAPD分子标记鉴定紫花苜蓿品种的反应体系优化

以中苜1号紫花苜蓿(Medicago sativa L.cv.Chongmu No.1)为研究材料,通过对RAPD-PCR反应退火温度、反应体系中的模板DNA、Taq聚合酶、Mg²⁺、dNTP、引物用量的梯度处理,分别对各项单因子进行优化。结果表明,当退火温度为37℃,总反应体积25μl时,各反应物适宜用量为模板DNA80ng,Taq聚合酶1.5U,Mg²⁺6.25×10^-5mmol,dNTP0.3×10^-5mmol,随机引物0.4×10^-5mmol,缓冲液KCl1.25×10^-3mmol,扩增结果清晰、稳定,并且在不同实验室扩增结果具有较好的一致性。     

结缕草属植物RAPD反应体系的优化

为建立一个稳定的结缕草属植物RAPD-PCR反应体系,以结缕草种源Z111(Zoysia japonica Steud.)为实验材料,研究了模板DNA浓度、Taq酶、mg²⁺浓度、dNTP浓度、引物及扩增缓冲液浓度等各个主要因素对结缕草RAPD-PCR反应的影响,并分别对各项单因子进行优化。结果表明:总反应体积为20×L时,各反应物的适宜用量分别为模板DNA浓度30 ng、Taq酶1.0U、mg²⁺2.5 mmol/L、dNTP 0.19mmol/L、引物0.5×mol/L、10×buffer2.0×L;5种、1变种共20份结缕草种源材料验证,显示该体系扩增条带多、清晰结果稳定,表明该体系是一个适合结缕草属植物RAPD-PCR反应的体系。     

山羊基因组DNA RAMP-PCR反应体系的建立

以山羊基因组DNA为材料,研究了MgCl2浓度、引物浓度、dNTP浓度、模板DNA用量、TaqDNA聚合酶用量对RAMP-PCR反应的影响.建立了一套适合山羊的最佳RAMP反应体系.反应总体积为25μL,MgCl2浓度2.0 mmol/L、引物为0.4 mmol/L、dNTP浓度为0.2 mmol/L、模板DNA为25 ng,TaqDNA聚合酶为1U.PCR反应程序为:94℃ 5 min;94℃ 30 s,40℃ 45s,72℃ 90s,30 Cycles;72℃ 10 min.     

基于Nc2和Nc5基因的新孢子虫PCR方法的建立

本试验筛选了新孢子虫病PCR检测的引物,运用《新孢子虫病检疫技术规范》(SN/T 3499-2013)对根据犬新孢子虫Nc2和Nc5基因设计的PCR引物进行了评价。此外,同时运用F1/R1、F2/R2和SN/T 3499 F/SN/T 3499 R共同对14份荷斯坦牛和19份西门塔尔牛全血DNA进行PCR检测,旨在筛选出特异性较好的引物,建立新孢子虫病PCR检测方法和了解当地不同品系牛患新孢子虫病的感染率。结果显示,3对引物分别扩增出105、128和231 bp目的片段,均与预期目的片段大小相符;其中,F1/R1与SN/T 3499 F/SN/T 3499 R的最低检测量相同,为19.9 fg/μL,F2/R2最低检测量为199 fg/μL,说明F1/R1和SN/T 3499 F/SN/T 3499 R引物的敏感性更好;运用F1/R1、F2/R2引物分别对19.9 pg/μL和199 fg/μL模板重复进行4次扩增,均出现了较明亮的扩增条带,证明两对引物重复性较好。33份血液样品共检出6份阳性DNA,阳性率分别为21.43%和15.79%,检出复合率为100%。以上结果说明F1/R1和F2/R2引物均可作为新孢子虫病PCR的诊断引物,本试验初步建立了新孢子虫PCR方法,同时初步了解了当地牛群中新孢子虫感染情况,为有效预防和控制新孢子虫病提供了科学的理论依据。     

抗蚜紫花苜蓿品系SSR体系的建立

以抗蚜紫花苜蓿品系HA-3为材料,研究了SSR反应体系中各个影响因素的浓度和用量,确定了适合苜蓿抗蚜基因定位的SSR体系:TaqDNA聚合酶0.2μL(5 U/μL),10×PCR Buffer(Mg2+Plus)2.5μL,dNTP Mixture 2μL(各2.5 mmol/L),引物1.5μL(10μmol/L),模板DNA 1μL,ddH2O16.3μL补足25μL。利用该体系进行扩增,所得谱带清晰、稳定、非特异带少。     

家蚕RAPD的扩增条件、重复性及遗传模型研究

发现当ＰＣＲ体系中Ｍｇ￣（２＋）为２ｍｍｏｌ／Ｌ、ｄＮＴＰｓ为１５０μｍｏｌ／Ｌ、Ｔａｇ酶为１Ｕ、引物为０．２μｍｏｌ／Ｌ时，以４０～４２℃退火可得到稳定结果；８％的ＤＭＳＯ对提高扩增特异性有利；分别从蚕卵、蚁蚕、５龄蚕丝腺、体壁及生殖腺、蛹、蛾中提取模板ＤＮＡ，其ＲＡＰＤ结果一致；以Ｃ＿（１０８）×大造为材料，发现其Ｆ＿１ＲＡＰＤ为共显性。     

破碎法提取家蚕微孢子虫孢子核酸的PCR检测灵敏度试验

蚕微粒子病检测技术是该病防治中的一项重要技术,本文在通过破碎法提高模板核酸获取量的基础上,对纯化家蚕微粒子虫孢子的PCR检测灵敏度进行了测试。提取核酸-模板稀释测试12对引物的灵敏度中有4对引物达到0．05粒（10^2粒/mL）、4对引物5粒（10^4粒/mL）、1对引物50粒（10^5粒/mL）、2对引物500粒（1...     

杂花苜蓿SRAP-PCR反应体系的正交优化

利用正交设计L₁₆(4⁵)对杂花苜蓿(Medicago varia Martin.)SRAP-PCR反应体系的5因素(Taq酶、Mg₂⁺、模板DNA、dNTP、引物)在4个水平上进行优化试验,旨在建立一套适用于苜蓿属(Medicago L.)种质资源的SRAP标记技术体系。结果表明:各因素对PCR反应结果都有显著影响,由F值可知,dNTP的量对反应结果影响最大,其次是Taq酶的量,模版DNA浓度的影响最小,各因素水平的变化对PCR反应的影响依次为:dNTP>Taq酶>Mg₂⁺>引物>模板DNA;筛选出各反应因素的最佳水平,建立杂花苜蓿SRAP-PCR反应的最佳体系(25μL)为:dNTP 2μL(0.20 mmol·L^-1),TaqDNA聚合酶0.3μL(1.50 U),Mg₂⁺3μL(1.50mmol·L^-1),上、下游引物各1μL(0.40μmol·L^-1),50ng模板DNA1μL,10×PCR buffer 2.5μL(不含Mg₂⁺)。这一优化体系的建立,为今后利用SRAP标记技术进行苜蓿属植物遗传图谱构建、遗传多样性分析及种质资源鉴定等研究奠定了技术基础。     

A multiplex polymerase chain reaction for discriminating Erysipelothrix rhusiopathiae from Erysipelothrix tonsillarum.

Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, and it causes great economic losses in Japan and worldwide. In meat inspection, it is very important to distinguish E. rhusiopathiae from other bacteria showing similar clinical signs of disease or similar bacterial characteristics. To distinguish E. rhusiopathiae from Erysipelothrix tonsillarum, 2 polymerase chain reaction (PCR) systems were combined. The primer sets ERY-1F and ERY-2R were designed to amplify 2210 base pairs (bp) of nucleotide sequence specific for E. rhusiopathiae chromosomal DNA, and the primer sets MO101 and ERS-1R were designed to amplify 719 bp of nucleotide sequence including a highly conserved region of genus Erysipelothrix 16S rRNA. Two fragments were amplified when E. rhusiopathiae was used as the PCR template using the primer sets, whereas a single fragment was amplified when E. tonsillarum was used as the template. No fragments were amplified when nucleic acid from other bacteria that cause clinical signs similar to swine erysipelas were used as the template. Moreover, 5 specimens collected from postinspected swine carcasses were diagnosed as E. rhusiopathiae using the PCR described in this study, in agreement with results of microbiological tests for the genus Erysipelothrix, whereas negative samples were negative both in conventional bacterial tests and by PCR. The detection limit of multiplex PCR ranged from 10(2) to 10(4) colony forming units per reaction tube for positive samples. These results suggest that this method is useful for screening of swine erysipelas in meat inspection centers.     

嗜线虫致病杆菌HB310菌株对家蚕的毒力及安全性评价

嗜线虫致病杆菌(Xenorhabdus nematophila)HB310菌株与小卷蛾斯氏线(Steinernema carpocapsae)互助共生,对多种寄生害虫具有杀灭作用。为了探讨该共生菌在蚕区大田农作物害虫防治上应用的可行性,研究了嗜线虫致病杆菌HB310菌液和蛋白毒素粗提物对家蚕的生物学安全性。结果表明:HB310菌液对家蚕的急性毒力很低,即使在处理后120h,仍然不能测定其LC50;家蚕对HB310菌液和蛋白毒素粗提物均表现出一定的拒食性,5龄幼虫对2.24×106CFU/mL HB310原菌液和0.57mg/mL杀虫蛋白粗提物的48h拒食率分别达到24.90%和23.05%;HB310菌液及蛋白毒素粗提物对家蚕各龄幼虫体重均有明显的抑制作用,并且随龄期而增大;从蚁蚕开始一直饲喂含HB310原菌液人工饲料的幼虫各龄历期均明显延长,存活率显著下降,到4龄全部死亡;幼虫取食含HB310原菌液人工饲料72h后改饲喂正常饲料,1龄和2龄的历期明显长于对照,但以后各龄历期、化蛹率、羽化率、蛹重和茧层量等各项生物学和经济性状指标均与对照无显著差异;1.12×104CFU/mL HB310菌液和0.11mg/mL蛋白毒素粗提物对家蚕的生长发育无显著影响。综合分析认为,HB310菌株对家蚕低毒性。     

家蚕精子介导转基因技术体系的优化

为了构建高效的家蚕转基因技术,对采用piggyBac转座载体的家蚕精子介导转基因的主要技术参数进行优化,结果表明外源DNA的稀释剂和注射方式对导入效率影响较大,G0代未整合的外源DNA在5龄期前基本被降解破坏。推荐的家蚕精子介导基因转移技术方案为:处女蛾交尾囊注射6～8μL以0.1×TE稀释的2 g/Lpig-gyBac转座子载体DNA后正常交配产卵,根据G0代5龄期及蛾期PCR检测标记基因为阳性的蛾区自交,G1代阳性个体自交以获得纯合后代。     

Development of a PCR Assay Based on a Single–Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene

Aeromonas caviae is a bacterial pathogen that causes various infectious diseases in both humans and animals. To facilitate its detection, we developed species-specific primer sets targeting polymorphisms in the gyrB gene for use in a PCR assay. The technique was able to detect 100% (29/29) of the A. caviae strains tested using either of two sets of primers (designated ACF1-ACR and ACF3-ACR), which produced 293-bp and 206-bp amplicons, respectively. Another set of primers (designated ACF2-ACR) yielded a 237-bp amplicon and exhibited 90% (26/29) positive results with respect to A. caviae. None of the primer sets exhibited cross-reactivity with 12 non–A. caviae isolates and 52 other non-Aeromonas bacteria. The detection limit using the ACF2-ACR and ACF3-ACR primer sets in pure culture was 1.6?×?10³ CFU/mL, or 6 CFU per reaction, whereas that of the ACF1-ACR primer set was 1.6?×?10⁴ CFU/mL, or 60 CFU per reaction. In the case of spiked Nile Tilapia Oreochromis niloticus, the sensitivity of all primer sets without enrichment was 1.8?×?10⁴ CFU/g, or 30 CFU per reaction. Primer set ACF3-ACR was the best for a PCR assay targeting the gyrB gene, and the PCR technique developed was rapid, specific, and sensitive for the identification of A. caviae.

Received December 2, 2014; accepted April 9, 2015     

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed,paraffin-embedded tissues

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.