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。     

牛卵形巴贝斯虫不同靶基因PCR检测方法的比较

旨在筛选出检测牛卵形巴贝斯虫特异、敏感的PCR方法。本试验以牛卵形巴贝斯虫18S rRNA、AMA-1和CCTη基因为靶基因进行PCR检测,从敏感性、特异性和临床检出率方面进行比较。结果显示,以18S rRNA基因的PCR方法敏感性最高,最小检出率为16 fg/μL;以CCTη为靶基因的PCR方法敏感性最低,检测量为1.6 pg/μL;而以顶膜抗原(AMA-1)为靶基因的PCR方法的最低检测量为160 fg/μL。三种靶基因均扩增不出牛瑟氏泰勒虫、牛巴贝斯虫和双芽巴贝斯虫基因片段。通过60份临床血液样本的检测结果表明,以18S rRNA基因设计引物的检出率最高,为30%(18/60),明显高于以AMA-1基因25%(15/60)和CCTη基因21.67%(13/60)。本试验为卵形巴贝斯虫病的诊断提供了更为敏感、特异的检测技术。     

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。     

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。     

犬巴贝斯虫实时荧光定量PCR检测方法的建立

为建立一种快速准确检测犬巴贝斯虫(Babesia cains,B.canis)的方法,试验根据GenBank中收录的巴贝斯虫18S rRNA序列设计一对特异性引物,对建立的基于SYBR GreenⅠ检测巴贝斯虫的实时荧光定量PCR方法进行了研究。结果表明:建立的实时荧光定量PCR方法可准确检测出10拷贝/μL的样本,灵敏度是常规PCR的1 000倍;该方法可以特异地检测巴贝斯虫,对6个对照组的检测结果均为阴性;批内和批间变异系数均低于5.00%;对豫西地区收集的21份临床样品进行检测,实时荧光定量PCR方法的阳性率为66.7%,普通PCR方法的阳性率为55.1%,血涂片法的阳性率为33.3%。说明该方法可用于临床疑似巴贝斯虫感染犬的早期检测和确诊。     

马梨形虫病双重PCR检测方法的建立

为了建立马梨形虫病双重PCR检测方法,试验根据Gen Bank发表的驽巴贝斯虫和马泰勒虫18S rRNA保守基因序列分别合成2对特异性引物,以核酸混合物为模板,优化反应条件建立马梨形虫病双重PCR检测方法,并检验该方法的特异性和敏感性。同时用该双重PCR检测方法对采自昭苏种马场的马疑似病例全血进行检测,并与镜检法和单重PCR法进行比较。结果表明:该双重PCR检测方法能对驽巴贝斯虫和马泰勒虫核酸扩增出大小为529 bp和789 bp特异性目的片段,而对双芽巴贝斯虫、环形泰勒虫、羊泰勒虫、牛巴贝斯虫核酸的扩增均为阴性;驽巴贝斯虫和马泰勒虫阳性DNA被稀释1×108倍时均能检出其相应目的片段;对采集的46份马疑似病例血样进行PCR诊断,其中驽巴贝斯虫感染率为30.4%(14/46),马泰勒虫感染率为41.3%(19/46);双重PCR检测方法与单重PCR方法的符合率为100%。说明建立的双重PCR检测方法具有一定的特异性和敏感性,可用于驽巴贝斯虫和马泰勒虫临床感染隐性病例的联合检测与鉴别诊断。     

牛双芽巴贝斯虫PCR检测方法的建立及初步应用

根据PCR技术原理,建立起了牛双芽巴贝斯虫病PCR检测方法,并测定出该方法的敏感性为108.66 fg。特异性试验表明,除牛双芽巴贝斯虫出现特异扩增带(295 bp)外,其他的虫株均未出现特异性扩增带。采用PCR检测方法,通过对35份牛血样的检测,测得牛双芽巴贝斯虫病的阳性率为28.57%(10/35),说明PCR方法敏感性强、特异性高,不失为牛双芽巴贝斯虫病临床诊断的好方法。     

套式PCR检测马巴贝斯虫

根据GenBank中马巴贝斯虫(Babesia equi)ema-1基因序列,设计合成内外2对引物,其中外引物扩增ema-1基因60～627nt间567 bp片段,内引物扩增ema-1基因259～488nt间229bp片段。从实验室感染马巴贝斯虫阳性马匹全血样本中提取DNA,采取2次扩增的方法,扩增到229bp特异性条带,建立了适合马巴贝斯虫快速检测的套式PCR方法。经重复性试验和特异性试验,结果显示,马巴贝斯虫阳性样本在229bp均出现条带,而驽巴贝斯虫、牛巴贝斯虫扩增结果为阴性。采用该方法对已知阴、阳性的16份马匹全血样品进行检测,有8份为阳性,与实际结果的符合率为100%。表明,所建立的方法具有较高的重复性和特异性,可用于马巴贝斯虫病的临床诊断、病料检测和分子流行病学调查。     

蜱传牛巴贝斯虫套式PCR检测方法的建立及应用

为建立一种快速、敏感检测牛巴贝斯虫的方法,本研究根据GenBank中登录的牛巴贝斯虫rap-1基因保守区设计2对特异性引物,经反应条件的优化,建立了牛巴贝斯虫套式PCR检测方法。结果显示,该方法可以特异地检测牛巴贝斯虫,而对双芽巴贝斯虫、牛环形泰勒虫和弓形虫的检测均为阴性;该方法的灵敏度可达1.3×10~1拷贝/μL,是牛巴贝斯虫荧光定量PCR检测试剂盒的10倍,是常规PCR的1 000倍。对50只扇头蜱和微小牛蜱DNA进行检测,套式PCR、牛巴贝斯虫荧光定量PCR检测试剂盒和常规PCR的阳性检出率分别为100.0%、72.0%和0。本实验建立的套式PCR检测方法适用于牛巴贝斯虫病的早期诊断和分子流行病学调查,为蜱传牛巴贝斯虫病的防控提供技术支持。     

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。     

Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay

We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina.     

Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in south Texas

The sudden death of several cattle infested experimentally with Rhipicephalus (Boophilus) microplus led to a clinical investigation into the reasons for the unexpected mortality. Microscopic evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all R. microplus strains received and propagated at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a well-conserved segment from the Babesia 18S ribosomal RNA gene followed by a nested PCR with Babesia species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. DNA from groups of 50 larvae was extracted using a rapid DNA preparation protocol, which consisted of grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5min. The assay sensitivity allowed for the detection of the equivalent of a single infected tick larva. R. microplus eggs were also analyzed, but yolk protein viscosity created inconsistent results with the crush and boil DNA isolation protocol, necessitating the use of a more extensive proteinase K digestion-based DNA purification method. We detected the presence of B. bigemina in all strains of R. microplus currently reared at the laboratory and 4 of 26 strains collected from infestation outbreaks in Texas by the U.S. Cattle Fever Tick Eradication Program.     

牛、羊布鲁菌多重PCR方法的建立与应用

根据布鲁菌属特异性基因BCSP31和布鲁菌种间特异性标志IS711插入序列,设计合成了3对引物,以牛种布鲁菌544A、104M和羊种布鲁菌16M基因组DNA为模板,通过优化反应条件,建立了可同时检测布鲁菌属、牛种布鲁菌和羊种布鲁菌的多重PCR方法。牛种布鲁菌可扩增出301和114 bp 2条带,羊种布鲁菌可扩增出301和253 bp 2条带,该方法对牛种布鲁菌544A和羊种布鲁菌16M混合DNA模板的最小检出量为100 pg,对大肠杆菌O157∶H7、小肠结肠炎耶尔森菌等15种参照菌的核酸扩增结果均为阴性。应用该方法对吉林省某牛场的106份粪便进行检测,虎红平板凝集试验作对照,结果PCR检测9份为阳性,且全为牛种布鲁菌阳性,对应的虎红平板凝集试验也为阳性。结果表明,建立的多重PCR方法具有良好的敏感性和特异性,为布鲁菌病的鉴别诊断提供了一种分子检测工具。     

Development and clinical evaluation of a PCR assay targeting the metalloprotease gene (mprA) of B. pseudomallei

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.     

Species-specific PCR assay for the detection of Babesia odocoilei

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.     

Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus

A single-step duplex polymerase chain reaction (PCR) technique and traditional microscopic examination of haemolymph smears were used to detect Babesia bigemina and/or Babesia bovis infection in engorged female ticks of Boophilus microplus recovered from calves raised in an endemic area of the State of Minas Gerais, Brazil. In the PCR amplification of tick-derived DNA, pairs of oligonucleotide primers specific for a 278-bp sequence from B. bigemina and for a 350-bp sequence from B. bovis were used conjointly. The microscopic examination of haemolymph revealed that 16.7% of the engorged ticks were infected with Babesia spp., although no significant differences (rho > 0.05) were found in the infection rate of ticks collected from calves of different age groups. PCR analysis showed that 77.8% of the engorged ticks whose haemolymph contained sporokinetes were infected with B. bigemina, 7.8% with B. bovis and 14.4% with both protozoan species. However, the PCR assay further revealed that, amongst the engorged female ticks whose haemolymph was apparently negative for the presence of sporokinetes, 15.6% were infected with B. bigemina, 2.2% with B. bovis and 10.0% with both species. The duplex PCR method is thus more efficient and sensitive than the microscopic assay and also permits facile identification of the protozoa species present in engorged female ticks.     

PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II

A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae.     

布鲁氏菌环介导等温扩增（LAMP）可视化检测方法的建立

应用环介导等温扩增(LAMP)技术建立了布鲁氏菌可视化快速检测方法。针对布鲁氏菌外膜蛋白OMP25基因保守区设计6条特异引物,反应前加入染料羟基萘芬蓝(HNB)作为LAMP扩增的指示剂,63℃恒温反应60min,根据HNB的颜色变化进行结果判定。分别评价所建立LAMP方法的特异性和灵敏性,并对60份牛布鲁氏菌病虎红平板凝集试验(RBT)阳性血清样本,经LAMP和B4/B5-PCR方法进行平行检测。结果显示,本方法最低检出限约为17fg布鲁氏菌基因组DNA。本方法特异性良好,布鲁氏菌反应管均出现特异性LAMP扩增反应,而猪大肠杆菌K99、巴氏杆菌C48-1、猪链球菌ST171、绿脓杆菌等对照组均未出现扩增。针对60份RBT阳性血清的平行检测结果显示,LAMP和B4/B5-PCR这两种方法间的结果符合率为85.0%。B4/B5-PCR检测为阳性的43份样品,经本方法检测全部为阳性;B4/B5-PCR检测为阴性的17份样品,经本方法检测,9份为阳性,8份为阴性。LAMP的敏感性高于B4/B5-PCR方法。实验表明,本文所建立的基于颜色判定的布鲁氏菌LAMP检测方法具有特异、灵敏、设备要求简单等特点,适用于基层兽医部门进行布鲁氏菌的快速检测。