羊泰勒虫PCR检测方法的建立和初步应用

利用羊泰勒虫18SrRNA基因的序列特点,设计合成种特异性引物,建立羊泰勒虫PCR检测方法,该方法能特异性扩增398bp的羊泰勒虫18SrRNA基因片段,而对羊巴贝斯虫、羊无浆体、牛环形泰勒虫和牛伊氏锥虫的基因组DNA没有扩增带出现。对羊泰勒虫基因组DNA的最小检测量为0.12fgDNA。通过检测124份临床样品,24份为羊泰勒虫感染阳性,其余为阴性。结果表明,建立的PCR检测方法具有极高的敏感性和特异性,可用于羊泰勒虫病和临床健康带虫羊的诊断。     

马泰勒虫病PCR检测方法的建立和应用

为寻求一种快速、有效的马泰勒虫 (Theileria equi,T.equi) PCR检测方法。基于马泰勒虫18S rRNA基因序列,在其V4高变区设计特异性引物Te-18F、Te-18R,通过PCR技术获得了531 bp的核酸片段。用该引物对马泰勒虫、马泰勒虫和驽巴贝斯虫混合样本、驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫基因组模板进行特异性试验。同时对马泰勒虫基因组模板进行不同浓度稀释后扩增,以便于确定试验的敏感性。用本试验建立的方法与常规显微镜镜检方法对45份马属动物血样进行检测。特异性试验显示,在被检测的9个样本中,只有马泰勒虫及马泰勒虫和驽巴贝斯虫混合模板中扩增出了符合大小的特异核酸片段。驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫的扩增结果均为阴性。灵敏度试验结果表明,PCR对马泰勒虫的扩增效率可达到10^-13。对本试验建立的PCR检测马泰勒虫方法评估结果显示,PCR对马泰勒虫的检出率为17.78%(8/45),显微镜镜检结果只有8.89%(4/45)两者的符合率为100%。本试验建立的马泰勒虫PCR检测方法不失,为一种好的检测方法。     

牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用

为建立特异、敏感、快速的二温式PCR诊断方法,根据牛环形泰勒虫裂殖子表面抗原(tams1)基因,设计了一对特异性引物,扩增出大小为154bp基因片段,经克隆、测序分析,与已知基因序列的相似性为96%。用建立的牛环形泰勒虫病二温式PCR诊断方法,对从新疆牛环形泰勒虫病流行地区采集的50份全血样品进行诊断,阳性率为88%,而血涂片检出的阳性率只有58%。经试验验证,该方法具有特异性高、敏感性强、重复性和稳定性好等优点。表明本试验所建立的二温式PCR诊断方法可用于牛环形泰勒虫病的临床诊断、隐性感染检测和流行病学调查。     

环形泰勒虫与牛无浆体双重PCR方法的建立

旨在建立快速检测环形泰勒虫和牛无浆体的双重PCR方法,调查吐鲁番地区这2种病原感染情况。根据NCBI库中上传的环形泰勒虫Spm2、Tams1基因和牛无浆体16S rRNA基因保守序列设计、合成3对特异性引物,使用PCR扩增并测序。根据Spm2和16S rRNA基因设计双重PCR并对反应体系及条件进行优化,对该方法进行特异性、灵敏性和重复性检测,建立一种双重PCR方法。结果显示,双重PCR方法特异性扩增出环形泰勒虫和牛无浆体相应目的片段,片段大小分别为853,351 bp,而驽巴贝斯虫、马泰勒虫、绵羊无浆体和伊氏锥虫均未扩增出条带。对环形泰勒虫Spm2、Tams1和牛无浆体16S rRNA基因序列进行系统发育分析,环形泰勒虫Spm2基因序列与印度株同源关系最近(MH844677)、Tams1基因序列与突尼斯株同源关系近(AF214899),牛无浆体16S rRNA基因序列与突尼斯株同源关系近(KY655808)。此方法能检测环形泰勒虫与牛无浆体最低浓度分别为2.9×10^-16,1.8×10^-19 g/μL。用该方法对临床60份牛血DNA进行双重...     

驽巴贝斯虫病PCR检测方法的建立和应用

根据驽巴贝斯虫(Babesia caballi)18S rRNA基因序列设计1对特异性引物,扩增出452 bp核苷酸片段,建立了检测驽巴贝斯虫病的PCR方法。敏感性试验结果表明,该方法最低能检出0.01 fg/μL驽巴贝斯虫DNA模板。特异性试验结果显示,在被检测的6个巴贝斯虫株中,仅驽巴贝斯虫株能扩增出特异性片段,马泰勒虫、双芽巴贝斯虫、莫氏巴贝斯虫、卵形巴贝斯虫、大巴贝斯虫的扩增结果均为阴性。对45份马属动物血样进行检测,本研究建立的PCR方法测得驽巴贝斯虫病的阳性率为26.67%(12/45),与显微镜检测方法进行了比较,结果显示PCR检测方法可显著提高驽巴贝斯虫的检出率。     

牛卵形巴贝斯虫巢式PCR诊断方法的建立及初步应用

根据GenBank上发表的牛卵形巴贝斯虫CCTη基因序列设计合成2对巢式PCR引物,建立牛卵形巴贝斯虫巢式PCR诊断方法,对该方法的最佳反应条件进行了筛选,并进行了特异性、敏感性及临床样本检测试验。结果表明,建立的巢式PCR方法外引物扩增牛卵形巴贝斯虫基因组片段的长度为1 008bp,内引物为537bp;该方法扩增不出牛瑟氏泰勒虫、弓形虫、犬新孢子虫基因组DNA;最低检测DNA含量为16fg;通过对46份临床样本的检测,该巢式PCR较常规PCR阳性检出率高8.7%。本试验为牛卵形巴贝斯虫病的诊断提供了一种更为特异、敏感的检测技术。     

羊泰勒虫病二温式PCR诊断方法的建立及初步应用

为建立特异、敏感、快速的羊泰勒虫病诊断方法。根据Gen Bank已报道的羊泰勒虫表面蛋白基因(AY274329)设计合成了1对引物,通过条件优化,建立了羊泰勒虫病二温式PCR诊断方法。该方法能扩增出335 bp的羊泰勒虫特异性基因片段,测序结果与已知基因序列同源性为100%。对羊泰勒虫基因组DNA的最小检测量为16 fg/μL,与新孢子虫、弓形虫、巴贝斯虫和瑟氏泰勒虫均无交叉反应。与普通PCR相比,二温式-PCR在敏感性方面无显著差异,但其反复升温降温时间消耗短。结果表明,二温式PCR诊断方法具有较高的特异性和敏感性,可用于羊泰勒虫病的快速诊断和流行病学调查。     

卵形巴贝斯虫与中华泰勒虫双重PCR检测方法的建立

为建立一种能快速对卵形巴贝斯虫(Babesia ovata)和中华泰勒虫(Theileria sinensis)同时进行检测的双重PCR方法。根据GenBank已报道的卵形巴贝斯虫AMA1基因和中华泰勒虫MPSP基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。结果显示：双重PCR可特异扩增出卵形巴贝斯虫和中华泰勒虫目的条带,片段大小分别为500 bp和986 bp。该方法具有较好的特异性,对卵形巴贝斯虫和中华泰勒虫的最低检出浓度为16 fg/μL。对采集的90份牛血液样本进行双重PCR检测,卵形巴贝斯虫阳性率为30%(27/90),中华泰勒虫阳性率为16.67%(15/90),混合感染率为10%(9/90)。结果表明,双重PCR方法可用于卵形巴贝斯虫和中华泰勒虫的快速诊断和流行病学调查。     

牛巴贝斯虫巢式PCR诊断方法的建立

根据GenBank发表的XJ-MSA-2c核苷酸序列(登录号:EU328267)设计的2对特异性引物MS-1、MS-2、MS-3以及MS-4,建立牛巴贝斯虫病巢式PCR快速检测方法。在特异性检测试验中,仅从MSA-2c质粒样本中扩增出622、350bp2条目的片段,与预期片段大小相符,而作为对照样本的双芽巴贝斯虫、牛环形泰勒虫、东方巴贝斯虫基因组DNA均无此扩增目的条带出现。第1次和第2次扩增的敏感性分别为1.75、1.75×10-2μg/L。在对46份全血的DNA样本巢式PCR和显微镜检测中,阳性检出率分别为34.8%(16/46)和23.9%(11/46)。结果表明,所建立的巢式PCR方法准确、敏感、特异,作为牛巴贝斯虫病的快速检测和小范围的流行病学调查,具有重要的临床意义。     

牛瑟氏泰勒虫ITS基因PCR诊断方法的建立

本试验根据GenBank上登录的牛瑟氏泰勒虫ITS基因序列(AY661522.1),应用Primer Premier 5.0和Oligo 6.31软件设计合成1对特异性引物,以牛瑟氏泰勒虫DNA为模板,建立了牛瑟氏泰勒虫ITS基因PCR诊断方法。该方法扩增片段大小为1020 bp,与参考序列的同源性为98%;建立的PCR方法与猪附红细胞体、犬新孢子虫和弓形虫均无交叉反应,最低DNA检出量为1.5 pg/μL;通过对60份临床样品的检测,并与血涂片方法进行比较,结果显示PCR方法具有特异、敏感等特点,适用于牛瑟氏泰勒虫的检测。     

Detection of Theileria ovis in naturally infected sheep by nested PCR

A nested polymerase chain reaction (PCR) for the detection of Theileria ovis in sheep using oligonucleotide primers designed from the small subunit ribosomal RNA (SSU rRNA) gene sequence of T. ovis from sheep in eastern Turkey is described. A 398-bp DNA fragment was specifically amplified from blood samples from sheep, naturally infected with T. ovis. No PCR products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesia spp. DNA using these specific primers. The sensitivity of the nested PCR for T. ovis, which was assessed showed that one infected cell in 10(7) sheep erythrocytes, equivalent to a blood parasitemia of 0.00001%, could be detected. This is more sensitive than examining 200 fields under light microscopy. In addition, of the 124 field samples obtained from sheep in eastern Turkey tested, 19.35% (24/124) were positive for the presence of Theileria spp. by microscopic examination compared to 54.03% (67/124) positive for T. ovis by nested PCR. The primer pairs described in this study will be useful for epidemiological studies on ovine theileriosis and for discrimination between T. lestoquardi and T. ovis infections in sheep.     

Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats

In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.     

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.     

A molecular survey of bovine Theileria parasites among apparently healthy cattle and with a note on the distribution of ticks in eastern Turkey

A survey of Theileria parasites in cattle in eastern Turkey was carried out using specific polymerase chain reaction. A total of 252 blood samples were collected from clinically healthy cattle between June and July 2004. Of 252 blood samples examined, 41 (16%) were positive for piroplasms by microscopy, whereas 114 (45%) were positive for the presence of at least one species of Theileria by PCR. The percentages of positive animals for Theileria annulata and benign Theileria species (Theileria sergenti/buffeli/orientalis) were 39% (99/252) and 7% (18/252), respectively. By allele-specific PCR examination of 18 field isolates which were positive for benign Theileria parasites, 8 samples were only amplified by B-type specific primers and 10 samples were amplified by both of the B and C-type specific primers, indicating a mixed infection with B and C-type of the parasite. None of the field isolates was amplified by I-type specific primers. Three samples were co-infected with T. annulata and benign Theileria parasites. Two of them which were infected with B-type parasite were also infected with T. annulata, the other sample which was infected both of B and C-type parasites was also infected with T. annulata. A total of 724 ixodid ticks were collected from the cattle. Hyalomma anatolicum anatolicum was the dominant species with 32% (230/724) in the region. H. a. excavatum, Boophylus annulatus and Rhipicephalus bursa represented 25% (183/724), 19% (140/724) and 15% (112/724) of the total number of ticks, respectively. R. sanguineus was the minor species and represented 8% (59/724) of the tick population.     

伪狂犬病病毒立即早期180基因的克隆及序列分析简

为了研究伪狂犬病病毒（pseudorabies virus,PRV）立即早期180基因（immediate early 180,IE180）及其功能,试验采用设计的特异性引物通过PCR法对IE180基因进行扩增,并成功构建了IE180克隆载体,经序列测定后,用DNAStar软件对该序列和已发表的7个PRV参考株的IE180基因核苷酸序列及推导的氨基酸序列进行比对分析。结果表明,HB-98株IE180基因编码区大小为4425 bp,编码1474个氨基酸,与GenBank中登录的参考株的IE180基因核苷酸同源性为98.4%～99.1%,氨基酸同源性为97.8%～98.4%。系统进化树分析结果表明,该毒株与毒株TNL(登录号：AF352564.2）的亲缘关系较近。     

牛环形泰勒虫裂殖子/梨浆虫表面抗原基因非疏水区的克隆及GST-P27重组蛋白的高效表达

A fragment about 696 bp was amplified by PCR technique with specific primers based on Tams1 gene sequence（AF214842） of Theileria annulata reported in GenBank. Then the target fragment was directionally cloned into pGEX-4T-2 expression vector, the recombinant plasmid DNA was cut by enzymes, and then sequenced.The result showed that the homology of the cloned Tams1 gene was 98%. The positive plasmid was transformed into BL21（DE3）, and then induced by IPTG. Soluble fusion protein whose expression level reached 1.39 mg/mL was obtained when it was induced with 0.5 mmol/L IPTG for 4 h at 37 ℃, and it was 53 ku. Western blotting showed that recombinant protein GST-P27 which was purified by Glutathione Sepharose 4B had the favorable reactionogenicity.