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本研究以质粒ｐＧＥＭ－７Ｚｆ（＋）为载体，对我国北方和南方疫区伊氏锥虫动基体株的ｋＤＮＡ小环进行了克隆，并以其中之ｐＴＫ０１１－Ｃ１为探针对不同地理区的动基体株和无动基体株的不同ＤＮＡ进行杂交。结果表明，我国北、南两大疫区动基体株的ｋＤＮＡ小环大小均约为１ｋｂ且均属Ａ型。ｐＴＫ０１１－Ｃ１只与动基体株的ｋＤＮＡ及ｔＤＮＡ杂交，不与其ｎＤＮＡ杂交，也不与无动基体株的任何ＤＮＡ及黄牛白细胞ＤＮＡ杂交。 相似文献
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伊氏锥虫微环DNA的PCR扩增 总被引：1，自引：0，他引：1
伊氏锥虫是动物伊氏锥虫的病原体，属动基体目原虫，动基体ＤＮＡ（ＫｉｎｅｔｏｐｌａｓｔＤＮＡ简称ｋＤＮＡ）是该目原虫所特有的一种非染色体ＤＮＡ。因此，对ｋＤＮＡ进行ＰＣＲ扩增可用于鉴定和检测伊氏锥虫。本文以５－ＣＡＡＣＧＣＡＡＡＧＡＧＴＣＡＧＴ－３’，５’－ＡＣＧＴＧＴＴＴＴＧＴＧＴＡＴＧＧＴ－３’为引物，对从伊氏锥虫直接抽提的总ＤＮＡ，ｋＤＮＡ以及ｋＤＮＡ基因重组子，经ＰＣＲ扩增后，在含有０．５μ 相似文献
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用长臂光敏生物素标记伊氏锥虫ｋＤＮＡ探针的研究王云飞，钟淑梅，周勇志（中国农科院上海家畜寄生虫病研究所）前言动基体ＤＮＡ（ｋＤＮＡ）是伊氏锥虫重要特征之一。应用同位素标记ｋＤＮＡ探针来检测伊氏锥虫，灵敏度高ｔ‘］。然而，同位素标记受实验条件限制，对于... 相似文献
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鸡传染性喉气管炎病毒中国王岗株gX基因的克隆及鉴定 总被引：4，自引：1，他引：3
以ｐＵＣ１９质粒为载体克隆鸡传染性喉气管炎病毒（ＩＬＴＶ）中国王岗株的ＤＮＡ，构建了鸡传染性喉气管炎病毒ＤＮＡ的ＫｐｎⅠＤＮＡ文库。参考ＩＬＴＶ－ＳＡ２株ｇＸ基因的核酸序列，设计并合成了分别为１２ｂｐ和１３ｂｐ的１对引物。以ＩＬＴＶ中国王岗株ＤＮＡ为模板，用ＰＣＲ方法特异性地扩增出０．８４ｋｂ的ＩＬＴＶ－ｇＸ基因片段。以地高辛标记该０．８４ｋｂ的片段为探针，经Ｓｏｕｔｈｅｒｎ杂交从ＩＬＴＶ中国王岗株ＫｐｎⅠＤＮＡ文库中筛选出３个含５．２ｋｂ外源ＩＬＴＶＤＮＡ片段的ｇＸ基因阳性重组子。经酶切分析、Ｓｏｕｔｈｅｒｎ杂交、ＰＣＲ检测和该片段部分酶谱分析表明，ＩＬＴＶ中国王岗株ＤＮＡ５．２ｋｂ的ＫｐｎⅠ片段无论是片段大小还是酶切图谱均与ＩＬＴＶ－ＳＡ２株完全相同，而且Ｓｏｕｔｈｅｒｎ杂交和ＰＣＲ检测均为ｇＸ阳性，证明其中含有完整的ｇＸ基因 相似文献
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牛传染性鼻气管炎病毒tk基因的克隆 总被引：2，自引：0，他引：2
根据牛传染性鼻气管炎病毒ＬＡ株的物理图谱及Ｃｏｐｅｒ株和ＬＡ标ｔｋ基因在基因组中的定位，将ＬＡ株ＤＮＡ ＨｉｎｄⅢＡ片段中的ＳａｌＩ－ＳａｌＩ亚片段、ＢｇｌⅢ－ＳａｌＩ双酶切亚片段分别克隆到载体质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ中，筛选出３个重组质粒ｐ＾ｔｋ－１，Ｐｔｋ－２和Ｐｔｋ－３，其外源片段大小分别为２．７ｋｂ，１．１ｋｂ和１．６ｋｂ。经酶切分析及同源核酸探针杂交试验表明，２．７ｋｂ ＳａｌＩ－ 相似文献
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Ｋ７００ｂｐ是高产蜜西蜂引物Ｋ（５’－ＣＧＧＣＣＣＣＴＧＣ－３’），通过ＲＡＰＤ－ＰＣＲ扩增出来的一个特有ＤＮＡ片段。然后将Ｋ７００ｂｐ制备成探针再与高、低产蜜西蜂的ＰＣＲ产物及它们的基因组进行杂交。结果Ｋ７００探针只与高产蜜西蜂的ＰＣＲ产物及其基因组杂交，证明Ｋ７００ｂｐ确实是高产蜜西蜂的一个特有ＤＮＡ片段。 相似文献
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从包含伪狂犬病病毒（ＰＲＶ）闽Ａ株ＢａｍＨＩ－７片段的重组质粒ｐＰＲ１２８中分离出含有完整糖蛋白ｇｐ５０基因的２．１ｋｂＤＮＡ片段，用ＫｐｎＩ和ＳｔｕＩ酶切后，将其酶切片段分别克隆到ｐＵＣ１９载体中，构建了２．１ｋｂ片段完整测序用质粒。对其序列进行分析，发现与文献报道结果一致，证明分离的ｇｐ５０基因是正确的。将包含ｇｐ５０基因的２．１ｋｂ和１．６ｋｂＤＮＡ片段分别插入带有痘苗病毒天坛株ＴＫ基因区段的ｐＧＪＰ－５质粒Ｐ７．５启动子的下游，构建了ｐＧＢＴ５０－３６和ｐＧＢＴ５０－Ｓ２２个嵌合载体。将嵌合载体通过磷酸钙共沉淀法转染预先感染ＴＫ＋痘苗病毒天坛株的人ＴＫ－１４３细胞或ＣＶ－１细胞，进行体内同源重组。经蚀斑纯化，在ＢｄＵＲ选择压力下，通过光敏生物素标记的探针杂交，获得带有ＰＲＶｇｐ５０基因的重组痘苗病毒。用ＥＬＩＳＡ检测，重组痘苗病毒有特异性ＰＲＶｇｐ５０抗原存在。 相似文献
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广州地区黑白花奶牛泌乳曲线数学模型的研究 总被引：2，自引：0，他引：2
本研究根据广州地区黑白花奶牛１１３个完整泌乳期的逐日产奶记录资料，研究了γ函数模型、多项式模型和回归模型的拟合和预报情况。结果表明：上述三个模型对广州地区黑白花奶牛泌乳曲线的拟合效果均不理想。因此，提出一个改进的多项式模型：＾ｙ＝ｋ＿０＋ｋ＿１ｔ＋ｋ＿２ｔ￣２＋ｋ＿３ｌｎｔ＋ｋ＿４ｌｎ￣２ｔ＋ｋ＿５ｅ（￣－１３７．５／ｔ）．加ｅ￣（－１３７．５／ｔ）这一项的目的是对奶牛中后期产奶量进行有效的修正。应用此模型有８０％样本拟合相对误差＜１０％，绝对误差＜５００ｋｇ，复相关系数＞０．９，全部样本均达Ｐ＜０．０１显著水平。 相似文献
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以大家畜和小鼠感染试验比较了我国南、北两大疫区伊氏锥虫株及南方疫区动基体株和无动基体株的致病力。结果显示，感染南方疫区动基体株和无动基体株的马属动物和牛的临床及血检变化极为相似，感染后者的小鼠平均存活时间比感染前者的长１６ｈ，但两虫株致病力无明显统计学差异。感染北方疫区动基体株小鼠的平均存活时间比感染南方株的长，与感染南方疫区动基体株组间差异显著（Ｐ＜０．０５），与无动基体株组间差异极显著（Ｐ＜０．０１）。提示北方疫区虫株致病力弱于南方疫区虫株。 相似文献
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应用质粒PTK探针鉴定锥虫的初步研究 总被引：1，自引：0，他引：1
用^32P标记质粒探针PTK1、PTK1.1和PTK1．2，对12株中国伊氏锥虫的斑点杂交试验显示，3个探针均能与8株具有正常动基体的伊氏锥虫杂交，而不与其余4株异常动基体伊氏锥虫杂交，对正常动基体株的敏感度为10^2虫体。探针PTK1亦能与马媾疫锥虫杂交，敏感度为10^2个虫体。但PTK1与布氏锥虫仅发生微弱的杂交反应．敏感度为10^5个虫体。试验表明伊氏锥虫株之间的kDNA微环是同源的，伊氏锥虫与马媾疫锥虫和布氏锥虫的kDNA微环存在着共同序列。 相似文献
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IntroductionCutaneous leishmaniasis (CL) is one of wobbling endemic disease in Iraq, that cause intracellular obligate protistan parasite returned to the genusLeishmania. This study is aimed to identify epidemiology of CL, detect the prevalence of Leishmania tropica and find the phylogenetic relationship.MethodologyThe current study was conducted in the main hospitals of Thi-Qar province-south of Iraq for period from November 2018 to October 2019. Nested-PCR was used to amplify kinetoplast minicircle fragments DNA.ResultsIt was recorded 247 clinical cases with CL, the infections of males were higher than females, while infection rate appeared gradual reduction with age progress. Furthermore, the most CL infections were as single lesions and occurred in December. The infections of upper limbs were high when compared with other body regions. The molecular diagnosis showed L. tropica was more frequently. DNA sequences of kDNA gene of L. tropica showed confirmative genetic detection of local isolates using NCBI-Blast data and phylogenetic tree analysis after comparison with global recorded isolates. The local L. tropica isolates showed genetically closed related to NCBI-Blast L. tropica with accession number AB678350.1. Generally, the analysis of kDNA nitrogen bases sequences showed that all of samples were consistent with those recorded at the NCBI.ConclusionThe kDNA minicircle sequences analysis results showed mismatching of the local isolates decrease whenever approached from the Iranian border. In addition, genetic heterogeneity diagnosis is important for detection of therapy, control and epidemiological studies. 相似文献
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Claes F Verloo D De Waal DT Majiwa PA Baltz T Goddeeris BM Büscher P 《Veterinary parasitology》2003,116(3):209-216
In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi. 相似文献
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Marcia Maria Pilatti Sidney de Almeida Ferreira Maria Norma de Melo Antero Silva Ribeiro de Andrade 《Research in veterinary science》2009,87(2):255-257
Four PCR assays for detection of Leishmania DNA in conjunctival swab samples were compared. All methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or seminested). Two methods (kDNA PCR-hybridization and kDNA snPCR) used primers targeted to the minicircles of kinetoplast DNA (kDNA) and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of ribosomal rRNA genes. kDNA PCR-hybridization was positive for 22/23 dogs (95.6%) and for 40/46 samples (86.9%), considering the right and the left conjunctivas. kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The ITS-1 nPCR and LnPCR were both able to detect the parasites in 17/23 dogs (73.9%) and 29/46 (63%) and 30/46 (65.2%) samples, respectively. The positivities of the kDNA based methods were significantly higher; however the choice of the best method will depend on the kind of information required with the diagnosis. 相似文献
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Basagoudanavar SH Rao JR Omanwar S Tiwari AK Singh RK Kataria RS Butchaiah G 《Acta veterinaria Hungarica》2001,49(2):191-195
A highly reproducible, dominant, monomorphic fragment of 473 base pair (bp) amplified from the genome of Trypanosoma evansi by arbitrary primer-polymerase chain reaction (AP-PCR) was labelled with digoxigenin and investigated for its potential as DNA probe. Dot-blot hybridisation of total genomic DNA with the probe proved useful in detecting bubaline, cameline and equine strains of T. evansi down to 10 pg of parasite template DNA. No cross-hybridisation was seen with Babesia bigemina, Theileria annulata and the bubaline host DNA. This probe may facilitate laboratory identification of T. evansi in developing countries, without the inherent risk associated with radioisotopes. 相似文献
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A TaqMan PCR assay was developed for the detection of Trypanosoma evansi. The assay targets the internal transcribed spacer 1 (ITS-1) region of rRNA. The ITS-1 region of eleven strains of T. evansi from widely separated geographical regions were sequenced and alignments compared. Primers and probe for the test were designed from these sequence data. The assay was tested using blood from infected rats and was found to be sensitive, detecting less than one genomic equivalent of T. evansi. The assay has been tested against 10 different species of trypanosomes found in native animals in Australia and did not detect any of these trypanosome species. Time course experiments using rats infected with T. evansi were performed to compare the TaqMan assay with the Haematocrit centrifugation test (HCT) and the mouse inoculation (MI) assay. The assay was more sensitive than the HCT but not as sensitive as the MI. The TaqMan assay has the ability to rapidly detect T. evansi and determine the number of organisms present in a blood sample from an infected animal. This is the first time a TaqMan assay has been developed for the detection of T. evansi. 相似文献
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为了确定中国伊氏锥虫各株的毒力强弱,对中国伊氏锥虫:安徽水牛株(AHB)、广东阳江水牛株(GDB_1)、广东水牛株(GDB_2)、广东马株(GDH)、广西骡株(GXM)、湖北骡株(HBM)、湖南水牛株(HNB)、江苏高邮水牛株(JSB_1)、江苏盱眙水牛株(JSB_2)、新疆骆驼株(XJCA)、云南水牛株(YNB)、浙江水牛株(ZJB)进行小鼠的毒力试验。以各组鼠死亡率和平均存活天数作为毒力强弱的主要依据。结果表明最强致死率为100%,最弱30%;致死所需时间平均为7.5 d～25 d。对小鼠的致病力强弱依次是:AHB>YNB>GDB_2>XJCA>HBM>HNC>JSB_1>GXM>GDB_1>GDH>JSB_2>ZJB。提示不同株的毒力差异显著,测定结果可为伊氏锥虫相关科学研究选株提供依据。 相似文献