应用低严格度-单引物聚合酶链反应对问号钩体中国参考株的基因多态性分析

Ｇ１、Ｇ２引物是对问号钩体具有特异性的引物。分别用Ｇ１或Ｇ２单引物对问号钩体中国参考株进行前４个低严格度循环的ＰＣＲ扩增，扩增带谱显示赖型、犬型、致热型、秋季型、澳洲型、临海型、乌尔夫型、溶血型为一类，而爪哇型、拜伦型、波摩那型、七日热型、巴叶赞型、塔拉索夫型、曼耗Ⅱ型则不与以上赖型等血清型钩体为一类，双曲钩体Ｐａｔｏｃ型及伊利尼细丝体伊利尼型的扩增带谱与问号钩体截然不同。应用苯酚法提取的高纯度钩体ＤＮＡ与ＳｉＯ２法提取的粗ＤＮＡ扩增结果一致。对钩体病患者血清标本进行ＬＳＳＰ－ＰＣＲ检测，可快速地对所感染钩体作出初步遗传学分类鉴定。     

早期钩体病患者血清钩体DNA聚合酶链反应和地...

We have detected and analysed the leptospiral DNA in serum of patients with early Leptospirosis from the epidemic area of China by PCR and DNA hybridization with Digoxigenin (Dig)-3-(2'-Spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)- phenyl-1,2-dioxetane (AMPPD) to develop a sensitive, specific and reliable technique for the early diagnosis of leptospirosis, and full satisfactory results have obtained. Fourteen serum specimens from patients with leptospirosis proven by blood culture and serological test were prepared according to Boom's methods for PCR test, and oligonucleotide primers, named G1 G2, were obtained from a genomic library of leptospira interrogans. PCR amplification with serum specimens was performed. Each cycle of amplification consisted of denaturation at 94 degrees C for 1 min, annealing at 55 degrees C for 1 min and elongation at 72 degrees C for 2 min. Each sample was subjected to 32 cycles. The amplified DNA were separated by electrophoresis with 2% agarose gel and hybridized with the homologous DNA probe labelling with Dig-AMPPD by means of Southern blotting. All of 14 samples revealed the presence of leptospira and the strong signals were visualized with homologous DNA probe hybridization by Southern blotting. Negative and positive controls appeared correctly. The DNA fragment generated from PCR amplification homologically hybridized with the DNA of 16 strains which are from Yasudas' genomic species and represent the different genomic groups of leptospires. The single recognized band (about 400 bps) from 6 out of the 16 strains has come out which are representative of the principal strains in Sichuan, China.(ABSTRACT TRUNCATED AT 250 WORDS)     

聚合酶链反应检测致病性钩端螺旋体微量DNA的研究

Leptospirosis is a severe zoonosis in the world. The methods for detecting leptospira are not sensitive and specific so far. The problems in early diagnosis and epidemiological identification of Leptospirosis remain unsolved. Two recombinant DNA fragments of serogroup Icterohaemorrhagiae, Leptospira were selected by repeated molecular cloning and screening in this study firstly. One of them can hybridize with the DNA of various serogroups of Leptospira interrogans; the other can only hybridize with the DNA of serogroup Icterohaemorrhagiae. After the nucleotides sequence analysis, from these 2 recombinant DNA fragments, 2 pairs of polymerase chain reaction (PCR) oligonucleotide primers were synthesized, named primer B 1, 2 and primer B 3, 4 PCRs were carried out with these 2 primers for detecting the microquantity (0.1 ng) of various serovars, serogroup of leptospires. All the DNA of Leptospira interrogans can be amplified by primer B 1, 2, and only the DNA of serogroup icterohaemorrhagiae, Leptospira reacted specially with primer B 3, 4. The DNA of non-pathogenetic Leptospira and some other microbes, however, had no amplification at all. This study is first reported at home and abroad. The results demonstrate that PCR is a very sensitive and specific technique of DNA amplification, which can be used as a powerful tool in the early diagnosis and epidemiological identification of leptospirosis.     

聚合酶链反应检测175例早期钩端螺旋体病患者...

We have developed a sensitive assay for leptospira, using the polymerase chain reaction (PCR). On the basis of the published nucleotides sequence of 23S rRNA gene from Leptospira interrogans serovar canicola strain Moulton, primers were chosen to produce an amplified fragment of 123 bp. Primer A: 5'GAT CTA ATT CGC TGT AGC AGG3' and primer B: 5'ACT TTC ACC CTC TAT GGT CGG3' Eight different svs. of Leptospira interrogans could all be detected by PCR, but the DNAs from L. biflexa. Leptonema bacteria, virus and human could not produce the specific amplified fragment. The assay detected approximately 10 fg of purified leptospiral DNA and 1 microliter serum of experimental animal. Positive results were obtained from simulated positive samples containing a single organism leptospiral DNA. The diagnostic test (proved by "gold standards": Clinical diagnosis; blood culture and MAT) showed that the sensitivity was 92.00%; the specificity 94.35%; the accuracy 92.54%; the positive predictive value 98.17%; the negative predictive value 78.13%; the positive likelihood ratio 16.25; and the negative likelihood ratio 0.0848. The diagnosis of early leptospirosis by using PCR may become a significant addition to diagnostic means.     

问号钩体血清型参考株16S rRNA基因的PCR－SSCP分析

利用１６ＳｒＲＮＡ基因引物扩增了我国致病性钩体１４群１５型参考株及赖型０１７株ＤＮＡ，通过非变性聚丙烯酰胺凝胶电泳，对各参考株的ＰＣＲ产物单链构象多态性进行了对比。结果显示在不同浓度凝胶及电泳条件下，１６株产物均有明显的２条单链，其中爪哇型、拜伦型、塔拉索夫型和曼耗Ⅱ型的国内参考株有相同的带型，而另１１型１２株的带型一致。此结果与Ｙａｓｕｄａ等（１９８７）和Ｒａ－ｍａｄａｓｓ等（１９９２）遗传学分种基本相符。     

钩端螺旋体16SrRNA逆转录聚合酶链反应

用ＳｉＯ＿２－高盐吸附法提取钩体ＲＮＡ，以问号状钩体１６ＳｒＲＮＡ基因引物对问号状钩体ｌａｉ型Ｌａｉ株和双曲钩体ｐａｔｏｃ型ＰａｔｏｃⅠ株的ＲＮＡ进行逆转录聚合酶链反应扩增。结果表明，采用本法对ＲＮＡ和ＤＮＡ同时检测时，在琼脂糖凝胶电泳中，可使肉眼检测水平提高约１００倍。     

66例早期钩体病患者血和尿标本的聚合酶链及生物素分子杂交…

采用国际通用引物Ｇ１Ｇ２对６６例发病在一周内钩体病患者的血和尿标本进行了ＰＣＲ检测及生物素－链霉抗生素蛋白碱酸酶体系标记的重组ＤＮＡ探针杂交分析。结果表明，ＰＣＲ技术结合生物素分子杂交分析，不仅排除了非特异性扩增，增加了可靠性，同时也提高了检测的敏感性（从７１．３％对８６．１％）；经统计学分析发现早期尿标本的ＰＣＲ是性率血标本ＰＣＲ检测阳性率没有统计学差别，由于尿标本易于收集和处理，所以对尿标本进     

聚合酶链反应检测175例早期钩端螺旋体病患者血清钩体DNA的研究

作者设计并合成了钩端螺旋体(简称钩体)犬群大型Moulton株23S rRNA基因的一对引物,即引物A_1 5'GAT CTA ATT CGC TGT AGC AGG~3'及引物B_1 5'ACT TTC ACCCTC TAT GGT CGG~3'用于聚合酶链反应(PCR)检测不同群型的问号状钩体,结果该引物不能使双曲钩体、细螺旋体和其它致病微生物及人白细胞DNA等扩增特异性片段,并用该对引物扩增早期钩体病患者和正常人及其它疾病患者的血清标本(以临床确诊、血培养及MAT阳性为金标准),检测结果表明:PCR诊断钩体病的敏感性为92.00％,特异性为94.35％,准确性为92.54％,阳性预测值为98.17％,阴性预测值为78.13％,阳性拟然比为16.25,阴性拟然比为0.0848。由此表明PCR扩增钩体235 rRNA基因,是早期钩体病诊断的一种敏感、特异、快速而简便的方法。     

问号赖型钩体重组质粒rpDJt表达蛋白P68免疫原性的研究

为构建一种新型钩体重组亚单位疫苗,顺号本ｒｐＤＪｔ基因的蛋白表达物Ｐ６８免疫动物,对其免疫原性进行研究。结果显示,在体外,Ｐ６８能与其特异性抗血清及赖型钩体死菌苗抗血清呈明显高效价Ａｇ－Ａｂ反应,在体内,能激起明显的Ｔ淋巴细胞转化,ＴＨ细胞活化,ＩＬ－２,ＩＬ－６活性增加。提示：ｒｐＤＪｔ表达蛋白Ｐ６８能激起机体强有力的Ｔ－Ｂ细胞同性特异性体液免疫反应,是致病性强毒力钩体重组亚单位疫苗好的候选抗     

赖型钩湍螺旋体基因库的构建及致病性钩端螺旋体DNA同源序列...

A gene bank of the main pathogen of pulmonary diffuse haemorrhage type leptospirosis (PDH), L. interrogans serovar lai strain 017, was first constructed with plasmid vector pUC9, which contained 610 recombinant clones and laid the foundation for further investigation of molecular characteristics of leptospires with strong virulence. Recombinant plasmids which have homological sequences of pathogenic leptospires were screened from the gene bank. A recombinant plasmid, designated pCX7, could detect 1.7 kb fragment of strain 017, 9.0 kb of strain 601 and 30.0 kb of strain 610 respectively without cross hybridization with nonvirulent leptospires such as L. biflexa strain Patoc I and Leptonema illini. pCX9, another recombinant plasmid, could detect 1.9 kb fragment of strain 017 and had no hybridization with other pathogenic or nonpathogenic leptospires. The results showed that the degree of homology between pathogenic and non-pathogenic leptospires was very low and the degree of homology was very high among the pathogenic leptospires.     

赖型钩体DNA重组质粒rpDJt及其表达蛋白P68对豚鼠的免疫？…

为了进一步鉴定赖型钩体ＤＮＡ重组质粒ｒｐＤＪｔ及其纯化表达蛋白质Ｐ６８例的免疫保护作用，采用随机、以盲和对照法对５０只豚鼠进行了３次和６个部位主动免疫接种，并于接种后３０天以强毒力、大剂量活钩体攻击。结果：Ｐ６８组存活率１００％（７／７）；ｒｐＤＪｔ组存活率７７％（１０／１３）；ｐＴ７－７组（无重组质粒）存活率２５％（３／１０）；全钩灭活疫苗组存活率９３％（１３／１４）。作者认为该质粒的表达蛋白质     

赖型钩体pDC38核酸序列测定及其与流感伤寒型钩体外膜蛋？…

为了探讨赖型钩体ｐＤＣ３８插入片段基因组ＤＮＡ编码、位置与ｏｍｐＬ１基因的关系,作者采用在流感伤寒型钩体外膜蛋白基因ｏｍｐＬ１首尾区段设计一对引物,以ｐＤＣ３８插一段作模板进行ＰＣＲ扩增、玫类似性匹配（ａｌｉｇｎｍｅｎｔ）。结果发现,ｐＤＣ３８含有２．７ｋｂ和３．０ｋｂ两个插入片段,３．０ｋｂ片段含有１个ｏｍ;Ｌ１基因全拷贝。结论：类似性匹配表明,ｐＤＣ３８和ｏｍｐＬ１相同碱基８６４个（９０％）,     

问号钩体（狭义）种特异探针重组质粒pCX7插入片段核苷酸序 …

为了建立敏感特异的鉴别致病性钩体菌侏的方法，用ＡＢＩ自动测序仪对问号钩体（Ｌ．ｉｎｔｅｒｒｏｇａｎｓｓｅｎｓｕｓｔｒｉｃｔｏ）种异探针ＤＮＡ重组质粒ＰＣＸ７，１．７ｋｂＰｓｔＩ部分插入片段进行了核苷酸序列测定。结果：该部分插入片段ｂｐ总数为１－５０１ｂｐ，其中可读框１个，序列为８７－３９３，共３０６ｂｐ。     

问号赖型钩体重组质粒rpDJt表达蛋白P68免疫原性的研究

为构建一种新型钩体重组亚单位疫苗，采用问号钩体ｒｐＤＪｔ基因的蛋白表达物Ｐ６８免疫动物，对其免疫原性进行研究。结果显示：在体外，Ｐ６８能与其特异性抗血清及赖型钩体死菌苗抗血清呈明显高效价Ａｇ－Ａｂ反应；在体内，Ｐ６８能激起明显的Ｔ淋巴细胞转化，ＴＨ细胞活化，ＩＬ－２，ＩＬ－６活性增加。提示：ｒｐＤＪｔ表达蛋白Ｐ６８能激起机体强有力的Ｔ－Ｂ细胞协同性特异性体液免疫反应，是致病性强毒力钩体重组亚单位疫苗好的候选抗原之一。     

高效液相色谱法测定菝葜中白藜芦醇的含量

目的建立高效液相色谱法测定菝葜中白藜芦醇含量的方法。方法采用KromasilTMC18(200mm×4.6mm,5μm)色谱柱;流动相:甲醇-水(40∶60);流速:0.8ml/min;检测波长:303nm。结果线性范围为0.0772~1.2352μg,r=0.9998,平均回收率为100.49%,RSD=1.03%。结论该法简便、快速、灵敏、重现性好,适用于菝葜药材的质量控制。     

以λgt11作载体构建赖型钩体基因文库及其重组质粒pDL121的…

为探讨赖型钩体抗原基因特点及其表达产物作为疫苗的可行性，采用问号赖型０１７株钩体经Ｈｈａ Ｉ和Ａｌｕ Ｉ限制性内切核酸酶部分消化，行ＥｃｏＲ Ｉ限制酶切位点甲基化，加上ＥｃｏＲＩ接头与去磷酸化的λｇｔ１１ＤＮＡ－ＥｃｏＲ１臂连接，体外包装后转染Ｅ．ｃｏｌｉ Ｙ１０９０，建成含２．１×１０＾４重组子的问号赖型０１７株钩体基因文库。用抗赖型钩体兔血清从上述基因文库中筛选出中阳性克隆，λＤＬ１２，亚克隆