检测中国对虾非包涵体型杆状病毒的PCR方法的建立

中国对虾非体型杆状病毒（ＰｃＮＯＢＶ）是中国大陆养殖的中国对虾（Ｐｅｎａｅｕｓｃｈｉｎｅｎｓｉｓ）暴发性流行病－－白斑征的主要病原,与台湾流行的ＰｍＮＯＢⅢ、泰国的ＰｍＮＯＢⅡ及日本的ＲＶ－ＰＪ（＝ＰＲＤＶ）有相似的致病特征、形态学及组织病理学特征。为了角ＰｃＮＯＢＶ与ＰｍＮＯＢⅢ两种地域分布邻近的毒株基因水平的关系,并建立早期、敏感、特异的检测技术,利用氯化铯密度梯度超速离心技术分离纯化了ＰｃＮ     

用PCR法直接快速筛查重组阳性克隆

应用ＰＣＲ法快速筛查插入有苯丙氨酸脱氨酶ｃＤＮＡ重组阳性克隆。方法：用于ＰＣＲ扩增的引物是位于载体ｐＥＴ２３ｂ启动子处的Ｔ７启动子引物和位于目的基因ＰＡＬｃＤＮＡ３’端终止密码ＴＡＡ处的引物。以灭菌吸头挑一单菌落加入ＰＣＲ体系扩增。结果：在筛查的３个克隆中,有２个阳性克降,并且插入方向正确,经ＤＮＡ序列测定得到进一步证实。结论：以ＰＣＲ方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方面,不     

应用PCR方法检测油菜菌核病菌对多菌灵的抗药性*

通过保守的寡核苷酸引物B1/B3扩增出油菜菌核病菌MBCHR和MBCS菌株的部分β-微管蛋白基因,结果发现编码的198位氨基酸由Glu(GAG)突变为Ala(GCG),表现高水平抗药性。根据MBCHR菌株的突变设计2个快速检测方法:第一种方法是根据MBCHR菌株197和198位密码子(GACGAG→GACGCG)形成ThaI酶切位点(3’CGCG 5’),将B1/B3的扩增产物874bp片段酶切成193bp和681bp片段,而MBCS菌株的PCR产物不被酶切;第二种方法用198位突变密码子作为3’末端碱基设计2个等位基因特异性寡核苷酸引物(ASO)用于“nested”PCR或直接从基因组DNA扩增。通过PCR扩增和ThaI酶切能直接检测油菜菌核病菌的MBCHR和MBCS菌株,所得结果与传统菌落直径法相吻合。     

应用PCR方法检测油菜菌核病菌对多菌灵的抗药性

通过保守的寡核苷酸引物B1/B3扩增出油菜菌核病菌MBCHR和MBCS菌株的部分β-微管蛋白基因,结果发现编码的198位氨基酸由Glu(GAG)突变为Ala(GCG),表现高水平抗药性。根据MBCHR菌株的突变设计2个快速检测方法:第一种方法是根据MBCHR菌株197和198位密码子(GACGAG→GACGCG)形成ThaI酶切位点(3’CGCG 5’),将B1/B3的扩增产物874bp片段酶切成193bp和681bp片段,而MBCS菌株的PCR产物不被酶切;第二种方法用198位突变密码子作为3’末端碱基设计2个等位基因特异性寡核苷酸引物(ASO)用于“nested”PCR或直接从基因组DNA扩增。通过PCR扩增和ThaI酶切能直接检测油菜菌核病菌的MBCHR和MBCS菌株,所得结果与传统菌落直径法相吻合。     

应用反转录—聚合酶链反应检测口蹄疫病毒

建立了一种适用于检测动物（猪、牛、羊）组织（肌肉、淋巴结、脊髓、扁桃体和蹄冠皮）和牛食道－咽部分泌物（Ｏ－Ｐ液）中的口蹄疫（ＦＭＤ）病毒核酸（ＲＮＡ）的反转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术。引物对是人工合成的两条２０ｍｅｒ寡核苷酸片段,它们的序列相应于ＦＭＤ病毒结构蛋白ＶＰ１基因后２／３区段,在４个血清型之间基本一致（保守）。ＰＣＲ产物经琼脂糖凝胶电泳检测。试验结果表明,ＲＴ－ＰＣＲ具有良好的     

RAPID DETECTION OF LISTERIA MONOCYTOGENES IN GROUND TURKEY BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

The aim of this study was to determine the prevalence of Listeria monocytogenes in packaged fresh ground turkey in Turkey using immunomagnetic separation (IMS) as a selective enrichment step in method and polymerase chain reaction (PCR). A total of 180 ground turkey samples were collected during a 1-year period. Thirty-two (17.7%) of the samples contained L. monocytogenes, 24 (13.3%) contained Listeria innocua, 7 (3.8%) had Listeria ivanovii and 5 (2.7%) had Listeria seeligeri by means of IMS-based cultivation method. A PCR assay was performed, based on hlyA gene-specific primers. In all L. monocytogenes isolates, hlyA gene was confirmed, indicating that the correlation between IMS-based cultivation and PCR methods was 100%. The results suggest that the prevalence of L. monocytogenes in ground turkey is relatively high in Turkey and that ground turkey should be produced under appropriate hygienic and technological conditions for the prevention of public health hazards.

PRACTICAL APPLICATIONS

Using fast and reliable methods to detect and identify foodborne pathogenic bacteria, including Listeria monocytogenes , is important to detect the risk of contaminated product and protect public health. In some ways it is time-consuming to isolate and identify the pathogenic microorganisms from food products using conventional techniques. Different methods or techniques can be used both for redounding the isolation chance and to gain time for this purpose. Immunomagnetic separation (IMS) and polymerase chain reaction (PCR) techniques are effective and rapid methods for separation, detection and confirmation of Listeria spp. from foods. In this study rapid, specific and sensitive IMS method was used to determine the prevalence of L. monocytogenes in fresh ground turkey and PCR technique was used for the verification of the L. monocytogenes isolates.     

聚合酶链反应快速检测胃组织和唾液中的幽门螺杆菌

针对幽门螺杆菌（ＨＰ）尿素酶Ａ基因设计一对引物进行聚合酶链反应,检测１株ＨＰ标准株和７株临床分离株均阳性,而４株其它肠道菌均阴性,特异性１００％。１０倍系列稀释试验表明敏感性达到１００ｐｇＤＮＡ水平。从３５例胃镜检查者取幽门旁组织块进行快速和常规尿素酶试验,细菌培养及ＰＣＲ检测,１５例ＨＰ阳性者ＰＣＲ检测也为阳性,其中７例阳性者有３例唾液ＰＣＲ检测为阳性,表明ＨＰ确存在于口腔中。本研究采用直接热裂解法处理临床标本,取其粗提物行ＰＣＲ,免除复杂的酚一氟仿抽提步骤,该法简便快速,且损失小,成功率高,在临床实验诊断中有推广价值。     

细胞培养中支原体污染的PCR检测

根据支原体16s rDNA序列,选择RemyTeyssou设计的三条寡核苷酸链,组成两套引物:P_(1-2a)能检测出细胞培养中常见的各种支原体,P_(1-2b)能检出无胆甾原体。反应可检出体系中10CFV的菌体。此法先用于对实验室人为污染支原体Vero细胞的检测,后与DNA 染色法和培养法比较,检测了49份生物样品,其中24份传代细胞,PCR检测的阳性率为58％,DNA染色法为42％,培养法为33％;三者的灵敏性比较,PCR可检出10~(-3)稀释度的阳性样品,高于其他两种方法。此PCR方法快速、灵敏、特异,适用于细胞培养中支原体污染的检测。     

用PCR检测临床血标本中人类细小病毒B19 DNA

用ＰＣＲ检测临床血标本中人类细小病毒Ｂ１９　ＤＮＡ杨洪江,张桦,林书祥,牛艾茹（天津市儿童医院儿研所,天津３０００７４）关键词聚合酶链反应（ＰＣＲ）,人类细小病毒Ｂ１９几种人类细小病毒中,只有Ｂ１９病毒是人类的病原体。由于该病毒不能在体外繁殖,获得该...     

用聚合酶链反应（PCR）检测支气管肺泡灌洗（BAL）液中的嗜肺军团杆菌

本文研究了由嗜肺军团杆菌的巨噬细胞感染性增效子（ｍｉｐ）基因设计的一对引物,用ＰＣＲ扩增嗜肺军团杆菌３、５、７、８血清型的４个标准菌株的特异ＤＮＡ序列,研究了用该引物扩增ＢＡＬ液中嗜肺军团菌特异ＤＮＡ序列的方法、灵敏度及特异性。结果表明：用上述引物扩增嗜肺军团菌４个标准菌株的ＤＮＡ,均可得到２０７ｂｐ的特异扩增产物,ＢＡＬ液中的军团菌经离心及裂解液裂解后,可直接进行ＤＮＡ扩增,当ＢＡＬ与液中军团菌量为２×１０３ＣＦＵ／ｍｌ时,即可检测出特异扩增带（电泳法）,除军团菌外,其它受试细菌均无此特异性扩增,用本法对４２例临床非典型肺炎患者的ＢＡＬ液进行嗜肺军团菌的检测,在４２份嗜肺军团菌培养均为阴性的ＢＡＬ液中,其中一例ＰＣＲ检测军团菌为阳性。本研究提示：用ＰＣＲ检测ＢＡＬ液中的军团菌是可行的,并有快速、灵敏、特异之忧点。     

多聚酶链反应技术鉴别肾综合征出血热病毒血清型的初步研究

肾综合征出血热(HFRS)为一组抗原性密切相关的布尼亚科汉坦病毒引起的急性传染病。在我国存在至少两种临床表现、动物宿主及流行特征截然不同的血清型别,即血清Ⅰ型(汉坦型)和血清Ⅱ型(汉城型)。这两型病毒间的血清学定型已有报道。近年来,除啮齿类动物外,从临床病人以及非啮齿类动物体内也分离到了HFRS病毒。同时出现两类型别毒株共存,以及从家鼠体内分离到野鼠型毒株或从野鼠体内分离到家鼠型毒株的复杂情形。为此,准确检定并鉴别不同来源毒株型别,将为深入了解其病原学、流行病学以及制定疫苗生产策略提供重要信息。     

REAL-TIME POLYMERASE CHAIN REACTION FOR DETECTION OF STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA IN PHARMACEUTICAL PRODUCTS FOR TOPICAL USE

Real-time PCR assays, based on LightCycler^TM hybridization probes technology, originally developed for detection of Staphylococcus aureus and Pseudomonas aeruginosa in clinical samples, were adapted to pharmaceutical products for topical use. After optimization experiments, the applicability of optimized PCR assays was assessed by testing 34 different pharmaceutical products for topical use in parallel with standard microbiological protocol according to European Pharmacopoeia. To reveal any problematic substances, which might inhibit PCR reaction, pharmaceutical products with as much different dosage forms as possible and of different composition were selected. Complete concordance between PCR and standard microbiological protocol results was obtained on a wide spectrum of pharmaceutical products. The adapted PCR assays can detect 1–10 CFU of both bacteria per gram or milliliter of pharmaceutical product in 26 h (including 24-h enrichment), whereas standard microbiological methods require 5–7 days. Real-time PCR assays proved to be efficient tools for rapid screening of S. aureus and P. aeruginosa in pharmaceutical products for topical use.