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1.
应用巢式聚合酶链反应（Nested PCR）-限制片段长度多态性 （restriction fragment length polymorphism, RFLP）方法对微小隐孢子虫（C．parvum）、安氏隐孢子虫（C．andersoni）和火鸡隐孢子虫（C．meleagrides）的鉴别进行了研究。结果显示C．parvum BOCC2、C．andesoni BOCC2和C．meleagrides CHCC1扩增产物片段大小分别为830bp、828bp和828bp，扩增产物分别经VspI酶切后形成3种不同的RFLP图谱，根据RFLP图谱可鉴别C．parvum、C．andersoni和C．meleagrides。本研究为我国隐孢子虫的分类和隐孢子虫病的分子流行病学研究打下了良好基础。 相似文献
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为了调查内蒙古部分地区规模化牛场奶牛隐孢子虫的感染情况及其虫种类型,试验在4个牛场采集了295份奶牛的粪样,从粪便样品中直接提取DNA进行套式PCR法检测,之后将其目的片段克隆至p GM-T载体上进行测序和同源性分析以鉴定虫种。结果表明:可通过套式PCR法从阳性粪便的DNA抽提物中扩增出目的条带,大小分别为800 bp和505 bp;对测得的序列和NCBI上已发表的安氏隐孢子虫(C.andersoni)18S rRNA基因序列进行BLAST比对分析,相似度为100%;采集样品的阳性率为14.9%(44/295)。说明套式PCR法可用于检测奶牛粪便样品中的隐孢子虫,且在一定程度上能够很好地反映出奶牛隐孢子虫的感染情况,并能有效鉴别其虫种。 相似文献
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目的对目前常用的两种检测隐孢子虫感染方法进行估价。方法取上海某牧场随机采集的20头奶牛粪便用改良抗酸染色法和巢式PCR(Nested PCR)法检测。结果两种方法均能检测出粪便中的隐孢子虫卵囊,改良抗酸染色法阳性检测率55%,Nested PCR法阳性检测率70%。结论:两种方法均能从感染隐孢子虫的奶牛粪便中捡出隐孢子虫卵囊,可根据需要选择。 相似文献
4.
采进口奶牛粪样200份,收集每份样品中的卵囊液,采用巢式PCR检测隐孢子虫(Cryptosporidium)18S rRNA基因,并用RFLP进行虫种鉴定,将目的片段克隆到pEGM-T easy vector,测序并进行同源性分析以进一步佐证虫种鉴定结果。结果表明,进口奶牛隐孢子虫阳性率为3.5%(7/200),514bp的目的片段不能被EcoT14 Ⅰ酶切。测序分析表明,获得的18S rRNA基因序列与NCBI上公布的微小隐孢子虫(C.parvum)相应序列同源性高达99.4%~100%,与安氏隐孢子虫(C.andersoni)同源性仅为91.1%。说明进口牛粪样中检测出的隐孢子虫为微小隐孢子虫。 相似文献
5.
PCR-RFLP鉴定隐孢子虫种类研究 总被引：15，自引：2，他引：15
为快速、准确鉴别人畜隐孢子虫种类，建立了巢式PCR扩增隐孢子虫18S rRNA基因的特殊区域，扩增片段测序结果表明：安徽牛源分离株(Cryptosporidium muris)781bp；北京鸡源分离株(C.baileyi)776bp；北京牛源分离株(C.muris)781bp；河南牛源分离株(C.muris)725bp；长春牛源分离株(C.muris)776bp；宁夏鸡源分离株(C.baileyi)725bp.该片段位于18SrRNA全序列271～1103bp之间。使用Ssp I限制性内切酶消化发现C.muris产生418～420bp和305～363bp两个片段，C.baileyi产生544～545bp和185～231bp两个片段。所检测的6个分离株可以显著区分为C.muris和C.baileyi两个种，所建立的PCR-RFLP可以有效鉴别隐孢子虫种类。 相似文献
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7.
巢式PCR检测隐孢子虫卵囊的研究 总被引：13，自引：0，他引：13
隐孢子虫病是一种重要的人畜共患原虫病。为了在临床样品中更准确、快速地检测隐孢子虫卵囊,从初步纯化的含有不同数量隐孢子虫卵囊的样品中和含有不同数量隐孢子虫卵囊的奶牛粪便中,直接提取DNA或用DNA纯化试剂盒对提取的奶牛粪便中卵囊DNA进行纯化之后用作起始PCR(Primary PCR)模板,以起始PCR的产物为模板进行巢式PCR(Nested PCR),用2对人工合成寡核苷酸分别作为两个PCR的引物,扩增片段大小分别为1325bp和820bp。优化了Mg2+浓度、引物浓度和dNTP浓度,并进行了特异性检验。建立的巢式PCR具有隐孢子虫属特异性,不仅扩增出新鲜样品DNA提取物中的目的片段,而且扩增出放置6年之久的DNA提取物中的目的片段。样品经过初步纯化之后,起始PCR和巢式PCR最低检测值卵囊分别为2 86×103个/ml和≤2 86个/ml;从含有隐孢子虫卵囊的奶牛粪便中提取DNA,尔后经过DNA纯化试剂盒纯化,其起始PCR和巢式PCR粪便中卵囊最低检测值分别为2 86×107个/g和≤2 86个/g。有望发展为试剂盒。 相似文献
8.
利用18S rRNA巢式聚合酶链反应(Nested PCR)-限制片段长度多态性(Restriction fragment length polymorphism,RFLP)鉴定吉林、大庆地区牛源隐孢子虫分离株.采取吉林、大庆地区断奶前犊牛粪便,提取DNA后经18S rRNA基因巢式PCR扩增,扩增产物测序后用Blast和MEGA4.0软件进行同源性和系统发育树分析.同时扩增产物分别用Ssp Ⅰ、Vsp Ⅰ和Mbo Ⅱ酶切后进行RFLP分析.通过18S rRNA基因PCR-RFLP分析和测序比对分析表明,吉林分离株包括2种隐孢子虫,分别为C.bovis和C.ryanae,大庆分离株包括3种,分别为C.boris、C.ryanae和C.andersoni. 相似文献
9.
安氏隐孢子虫PCR检测方法的建立 总被引：1，自引：1，他引：1
经BLAST检索,以HSP70基因设计一对引物(5'-CAATCGAATTGGATTCTTTGTC-3'和5'-CACCTTCAAAT-ACTTGAATAAGT-3')对奶牛安氏隐孢子虫进行了PCR试验.结果显示所建立的PCR检测方法只能特异扩增隐孢子虫GD株DNA,而对照样本如微小隐孢子虫、弓形虫、圆孢子虫、纤毛虫、肝片吸虫、血矛线虫、莫尼茨绦虫、牛粪便以及大肠杆菌均为阴性;通过对6个浓度梯度的虫体DNA进行PCR反应,结果表明当样本中含有445个隐孢子虫卵囊的DNA时,即可扩增产生清晰可辩的条带.测得该序列长度为494bp,序列分析为牛型C.andersoni.表明该引物能特异扩增C.andersoni,敏感性较高,适合于奶牛安氏隐孢子虫的检测. 相似文献
10.
目的为了建立一种快速、特异、灵敏检测动物源性食品中弓形虫的技术。方法根据原虫rDNA的部分序列,找出弓形虫和新孢子虫共同保守DNA片段,设计套式PCR两对引物,以UltraPureTM基因组DNA快速提取试剂盒提取弓形虫和新孢子虫DNA为模板,初步建立了检测两种虫体的Nested PCR技术。将纯化的Nested PCR产物成功地克隆到pGEM-T-easy载体中,经鉴定、测序并进行同源性分析。结果Nested-PCR的外、内引物对两种虫体rDNA基因均能进行扩增,长度分别在800~900 bp、400~500 bp之间。内引物扩增DNA序列与公布的同种虫体DNA序列同源性较高,弓形虫和新孢子虫分别达99.1%、97.2%,但它们两者之间差异较大,同源性仅为86.6%。用软件寻找能区别两者基因序列差异的酶切位点,挑选内切酶进行RFLP实验,结果表明新孢子虫NestedPCR扩增片段能被Vsp1酶切。同时进行该分子检测技术的特异性和敏感性试验,实验证明该Nested PCR能对弓形虫和新孢子虫rDNA基因进行特异性的扩增,而对其它原虫基因未能扩增出任何片段;该Nested-PCR能检测出100个弓形虫速殖子/g猪肉。结论本实验建立Nested PCR检测方法不仅可用于检测动物性食品中弓形虫,而且能明确区分弓形虫和新孢子虫。 相似文献
11.
Bakheit MA Torra D Palomino LA Thekisoe OM Mbati PA Ongerth J Karanis P 《Veterinary parasitology》2008,158(1-2):11-22
Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates. 相似文献
12.
Dung samples were collected from dairy calves of south Indian states viz., Andhra Pradesh, Karnataka, Kerala, Tamil Nadu and union territory, Puducherry and are subjected to nested polymerase chain reaction (PCR) targeting 18S rRNA gene for detection of Cryptosporidium infection. Of the 459 dung samples screened 182 were found positive with a prevalence of 39.65%. Highest prevalence of Cryptosporidium was observed in Puducherry (86.67%) and lowest in Kerala (17.65%). Genotyping by PCR-restriction fragment length polymorphism (RFLP) and sequence analysis revealed the presence of all the four major Cryptosporidium species of cattle viz., Cryptosporidium andersoni, Cryptosporidium ryanae, Cryptosporidium parvum and Cryptosporidium bovis. C. andersoni was widely distributed in calves of Tamil Nadu, Karnataka and Puducherry whereas in Andhra Pradesh C. ryanae was the major species. Of the 64 samples subjected to PCR-RFLP, 39 (60.94%) could be classified as C. andersoni, 18 (28.13%) as C. ryanae, 4 (6.25%) as C. parvum and 3 (4.69%) were confirmed as C. bovis. The results were also confirmed by sequencing of 19 Cryptosporidium DNA samples. 相似文献
13.
Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States 总被引：6，自引：0，他引：6
The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves. 相似文献
14.
Recent studies have identified the novel, host adapted Cryptosporidium bovis and the deer-like genotype in dairy cattle from farms in the United States, China, India and Europe. This novel species and genotype appear to be more prevalent in older, post-weaned dairy cattle than previously thought. However, little information is available on their prevalence in beef cow-calf operations. In the present study, we determined the prevalence of Cryptosporidium species in 98 calves (6-8 months old) and 114 cows (>2 years old) in seven beef cow-calf herds in western North Dakota. DNA was extracted from fecal samples and Cryptosporidium spp. were identified by amplification of the 18S rRNA gene followed by sequencing or RFLP analysis. All seven herds tested positive for Cryptosporidium. Overall, 43/212 (20.3%) animals were positive. Only five of these positives were from cows. C. bovis, the deer-like genotype and C. andersoni were identified in 9.4, 6.6 and 1.4% of animals sampled, respectively. C. parvum was not identified in any of the positive samples. C. bovis, the deer-like genotype and C. andersoni were detected in 6/7, 5/7 and 2/7 herds, respectively. C. bovis and the deer-like genotype were primarily detected in calves, while C. andersoni was only detected in cows. Six isolates could not be typed. These results show a relatively high prevalence of C. bovis and the deer-like genotype in 6-8-month-old beef calves compared to cows older than 2 years in the seven herds studied. 相似文献
15.
Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara (Hydrochoerus hydrochaeris) from Brazil 总被引：1，自引：0，他引：1
A total of 145 capybara (Hydrochoerus hydrochaeris) fecal samples from the state of S?o Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C. parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C. parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife. 相似文献
16.
Maikai BV Umoh JU Kwaga JK Lawal IA Maikai VA Cama V Xiao L 《Veterinary parasitology》2011,178(3-4):241-245
Despite numerous molecular epidemiologic studies of cryptosporidiosis in dairy cattle in industrialized countries, there are very few studies on the diversity and public health significance of Cryptosporidium species in native cattle in developing countries. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA gene was used to detect and identify Cryptosporidium spp. in 194 fecal specimens from 2 to 365 days old calves in 20 White Fulani and Sokoto Gudali herds in Nigeria. Thirty one (16.0%) of the specimens were positive for Cryptosporidium. Restriction digestion of the PCR products showed the presence of Cryptosporidium bovis (7.2%), Cryptosporidium ryanae (4.1%), Cryptosporidium andersoni (2.5%), and concurrent occurrence of C. bovis and C. ryanae (1.5%), and C. bovis and C. andersoni (0.5%). There were no significant differences (p>0.05) in Cryptosporidium infection rates by sex, herd location, management system, breed of calves, or fecal consistency. However, calves 180 days or younger had a higher infection rate of Cryptosporidium than older calves (p=0.034). Likewise, younger calves also had higher occurrence of C. bovis and C. ryanae (p=0.022). The absence of zoonotic Cryptosporidium parvum in the calves studied suggests that native breeds of cattle may not be important in the transmission of human cryptosporidiosis in Kaduna State, Nigeria. 相似文献
17.
以隐孢子虫(Cryptosporidium spp.)18S rRNA为靶基因,通过巢氏PCR检测发现在采集的101份新鲜粪便样品中18份样本为阳性。不同地区羊场的隐孢子虫感染率分别为：李集镇36%(18/50),新安镇0%(0/30)、堆沟港镇0%(0/21)未检出隐孢子虫。但通过对4羊场的分析发现,有2个羊场(50%)为隐孢子虫感染阳性,且不同的羊场感染率差异显著,因此单纯的以地区来评价隐孢子虫的感染率,是值得商榷的。山羊隐孢子虫的感染率为33.3%,湖羊隐孢子虫的感染率为2%。2～6月龄的育肥羊隐孢子虫的感染率为36%,6～10月龄的育成羊(0%)。对检测为阳性的样品进行了隐孢子虫18S rRNA基因片段序列分析,发现18个样品全部为肖氏隐孢子虫(Cryptosporidium xiaoi),不存在泛在隐孢子虫(Cryptosporidium ubiquitum)。在检测隐孢子虫感染阳性的1个山羊场和1个羊湖羊场均存在肖氏隐孢子虫感染,不存在泛在隐孢子虫,更未发现肖氏隐孢子虫和泛在隐孢子虫的混合感染。目前的数据提示肖氏隐孢子虫对2～6月龄山羊(33.3%)和湖羊(2%)具有更高的... 相似文献