实时荧光PCR检测牛边缘无浆体方法的建立及应用

根据牛边缘无浆体表面蛋白4的保守基因序列设计特异引物AMOC9/AMOC5、AMOC10/AMOC12和特异性探针MP,首次建立了牛边缘无浆体的实时荧光PCR检测方法,检测DNA的最低限度为200 fg。对中央无浆体、绵羊无浆体、牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫和伊氏锥虫进行检测,无荧光检测信号。本研究用所建立的方法检测采自江苏和哈尔滨的180份抗凝血（奶牛和肉牛）,其阳性率为8.9%。结果表明,建立的实时荧光PCR检测牛边缘无浆体的方法具有较高的特异性和敏感性,可用于牛边缘无浆体病的流行病学调查、检疫和监测。     

Detection and identification of Salmonella Typhimurium in bovine diarrhoeic fecal samples by immunomagnetic separation and multiplex PCR assay

The aim of this study was to use the immunomagnetic separation (IMS) test plus a multiplex polymerase chain reaction (m-PCR) assay to detect Salmonella at genus level and also for the identification of Salmonella enterica serovar Typhimurium in bovine diarrhoeic fecal samples. In all, 400 bovine diarrhoeic fecal specimens were examined by conventional bacterial culture, IMS, and m-PCR. For m-PCR assay, four set primers were selected: 139-141, specific for inv-A gene of Salmonella spp and the RfbJ, FliC and FljB, specific for the rfbJ, FliC and fljB genes of Salmonella Typhimurium or other Salmonella serovars with similar antigenic properties. Thirty-three (8.25%) out of the 400 fecal samples were culture positive for Salmonella serovars. Of these, 66.7% (22 of 33) were Salmonella enterica serovar Typhimurium, and 9.1% (three of 33) were serovar Dublin. In the IMS + m-PCR, four amplified product (663, 526, 284 and 183 bp) were found in all specimens that had serovar Typhimurium (4,5,12:i:1,2), they corresponded, respectively, to the rfbJ, fljB, inv-A and Flic genes of this serovar. In serovar Dublin (1,9,12:g,p:-), Georgia (6,7:b:e,n,z(15)) and, Enteritidis (1,9,12;g,m:-) only one PCR product (284 bp) was amplified from the inv-A gene. In serovars Augustenborg (6,7:i:1,2) and Lindenburg (6,8:i:1,2) three positive bands (526, 284 and 183 bp) were amplified corresponding to the fljB, inv-A and Flic genes, respectively. In serovar Virchow (6,7:r:1,2) two amplified products (284 and 526 bp) from the inv-A and FliC genes were observed. In serovar Gloucster (1,4,12(27):i:1,w) three fragments (183, 284 and 663) from the FliC, inv-A and, rfbJ genes respectively, were observed. In the positive control as expected, four PCR products were amplified corresponding to the FliC, inv-A, fljB and rfbJ genes, respectively. In conclusion, the results of this study showed that detection of Salmonella at genus level with universal ST139-141 primers and identification of Salmonella Typhimurium by using specific primers of O4, H(2):1, 2 and H(1) antigens can potentially permit to more readily evaluate fecal and other types of samples for the presence of these organisms. Compared to bacteriological culture the combination of IMS and m-PCR resulted a faster method for the detection and identification of Salmonella at genus and serovar level by using of universal and specific primers.     

用PCR和PCR-RFLP方法检测和鉴定进口海鱼中的异尖线虫幼虫

应用PCR和PCR-RFLP方法对厦门口岸进口的各种海鱼中的异尖线虫幼虫进行检测鉴定。结果在带鱼、竹荚鱼、金线鱼、大眼鲷和白姑鱼等海鱼中发现了简单异尖线虫、典型异尖线虫、对盲囊线虫和针回线虫等4种异尖线虫。带鱼和竹荚鱼的异尖线虫感染率达100%,竹荚鱼异尖线虫感染强度最高,白姑鱼感染率和感染强度相对较低。少数海鱼同时感染2种以上异尖线虫,从带鱼中同时检出简单异尖线虫和针蛔线虫,从竹荚鱼中检出简单异尖线虫、对盲囊线虫和典型异尖线虫。内切酶实验结果表明,应用限制性内切酶HinfI有效鉴别上述4种异尖线虫。     

用POR和POR-RFLP方法检测和鉴定进口海鱼中的异尖线虫幼虫

应用PeR和PCR—RFLP方法对厦门口岸进口的各种海鱼中的异尖线虫幼虫进行检测鉴定。结果在带鱼、竹荚鱼、金线鱼、大眼鲷和白姑鱼等海鱼中发现了简单异尖线虫、典型异尖线虫、对盲囊线虫和针回线虫等4种异尖线虫。带鱼和竹荚鱼的异尖线虫感染率达100％,竹荚鱼异尖线虫感染强度最高,白姑鱼感染率和感染强度相对较低。少数海鱼同时感染2种以上异尖线虫,从带鱼中同时检出简单异尖线虫和针蛔线虫,从竹荚鱼中检出简单异尖线虫、对盲囊线虫和典型异尖线虫。内切酶实验结果表明,应用限制性内切酶UinfI有效鉴别上述4种异尖线虫。     

Detection of pathogens in ovine and caprine abortion samples from Sardinia, Italy, by PCR.

During 2003-2005, 399 abortion samples (315 fetuses and 84 placentae) were collected from 107 ovine and caprine farms in northern Sardinia. Tissues from aborted fetuses and placentae were examined by PCR assay to detect DNA from Coxiella burnetii, Chlamydophila abortus, Salmonella enterica Serovar abortusovis, Toxoplasma gondii, and Neospora caninum. The DNA from at least 1 of these 5 infectious agents was amplified in 41% of ovine fetuses, while only 17% of the caprine fetuses yielded a positive amplification result for at least 1 of the 5 agents. Out of a total of 366 ovine aborted samples, T. gondii DNA was detected most frequently (18.1% of fetuses and 13.1% of placentae), followed by S. abortusovis (13% of fetuses and 14.4% of placentae), C. burnetii (10.9% of fetuses, of 9.2% placentae), C. abortus (2.4% of fetuses, 6.5% of placentae), and N. caninum (2% of placentae). In 33 fetuses and 9 placentae, the simultaneous presence of pathogens with different associations was detected. Out of a total of 31 caprine aborted samples, T. gondii was detected most frequently (13% of fetuses and 25% of placentae), followed by C. abortus (12.5% of placentae), C. burnetii (12.5% of placentae), and N. caninum (8.6%).     

Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods

We examined nasal swab and lung homogenate samples collected from pigs experimentally and naturally infected with Mycoplasma hyopneumoniae for the detection of M. hyopneumoniae by the nested PCR (nPCR) and culture methods. In the 23 experimentally infected pigs, M. hyopneumoniae was commonly detected in nasal swabs by the nPCR and culture methods at 4 weeks after inoculation, and there was a significant correlation (P<0.01) between the titers of viable organisms in nasal swabs and in lung homogenates in the experimentally inoculated pigs. In the naturally infected pigs, on the other hand, discrepancies in detection were found between nasal swab and lung homogenate samples in 17 of 36 cases, although the presence of gross lung lesions correlated relatively well with the detection of organisms from the samples. Our results indicated that the diagnosis of mycoplasmal pneumonia by nPCR in individual pigs with nasal swabs is reliable under these experimental conditions. At present, nPCR with nasal swabs should only be used for monitoring the disease status at the herd level under field conditions.     

Detection of Coxiella burnetii in placenta and abortion samples from British ruminants using real-time PCR

A real-time PCR was developed to detect Coxiella burnetii (the cause of Q fever) in ruminant placentas and aborted fetuses. Primer and probe sets previously developed for human tissue studies were used to target the insertion sequence IS1111 gene for C burnetii. The assay was highly sensitive, with a limit of detection of 10 copies of template, theoretically equating to a single bacterium, and did not cross-react with a panel of other bacteria. To determine sensitivity on field samples submitted for the diagnosis of abortion, results using the IS1111 PCR assay were compared with a com1 PCR assay. When applied to ruminant abortion material, including placental cotyledons and fetal samples, the IS1111 and com1 assays yielded positive results in 23 (25 per cent) of 93 and 19 (20 per cent) of 93 samples, respectively. One infected goat herd was monitored for 31 months: 57 (92 per cent) of 62 placental cotyledon samples from aborting and non-aborting goats, and 10 (30 per cent) of 33 fetal samples were positive by the IS1111 PCR assay.     

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

A PCR based method for the identification of equine influenza virus from clinical samples.

In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza virus (A2). We show that the assays are specific for the target genes chosen, and display sensitivity similar to virus isolation. The NP assay detects a variety of different influenza subtypes, whereas A1 and A2 assays are specific for influenza subtypes H7N7 and H3N8, respectively. Sequencing of amplicons obtained in the A2 assay yields information on antigenic regions of the haemagglutinin molecule, and use of this procedure in the routine surveillance of equine influenza will enable tentative characterisation of circulating viruses despite difficulties in isolating field strains of the H3N8 subtype. The A1 assay will be useful in ascertaining whether viruses of the H7N7 subtype still circulate amongst horses, or whether these are extinct.     

动物粪便沙门菌PCR检测技术的研究

为了建立一种动物粪便样品中快速、特异、敏感的沙门菌检测方法,根据沙门菌肠毒素stn基因设计一对引物,对不同血清型的沙门菌和非沙门菌进行PCR检测,并对该PCR方法进行反应的灵敏度、模拟粪便样品的最低检出限测定及粪便样品的检测。运用该方法对250份粪便样品进行检测,同时用传统方法进行验证。结果表明,设计的引物特异性好,能专一性扩增出约260 bp条带;该引物灵敏度高,能进行有效检测的核酸最低起始量为43.85 pg,纯菌检测灵敏度达1 cfu/mL。接种沙门菌到粪便样品中,当粪便样本中菌量达103cfu/mL以上时,不需要增菌,沙门菌可立即检出,增菌后检出限可以达到1 cfu/mL。PCR检测250份样品共检出18份阳性,同时传统细菌培养方法证明结果正确。该方法可用于粪便样品的检测。     

Detection and identification of banned processed animal protein in feedingstuffs by microscopic and PCR methods

The aim of the study was to present the results of comparative evaluation of the usefulness of PCR and microscopic methods for the detection of Processed Animal Protein (PAP) in feedingstuffs. In the validation study, the limit of the detection for PCR was determined on 0.05% for beef, 0.1% for pork and 0.2% for poultry meat and bone meal (MBM). Among 62 doubtful samples of feedingstuffs examined by microscopic method 41 (66.13%) were found as positive. Based on the results obtained with the use of the microscopic and PCR methods it is possible to state that the molecular biology methods can, at present, be used as a supplementary method in PAP detection.     

应用PCR技术检测鸽源性成分

根据鸽子线粒体DNAD-loop环基因中的保守序列,用Primer5.0设计针对鸽的特异性扩增引物。通过聚合酶链式反应从鸽样品中得到约150bp的特异条带。通过测序对扩增产物进行验证,鸽组织的PCR产物序列与基因库中检索到的相应序列相吻合。该实验建立了检测鸽动物源性成分的PCR方法,其检测灵敏度为0.1%。     

实时荧光定量PCR检测布鲁菌方法的建立

旨在建立实时荧光定量PCR检测布鲁菌的方法。经选取高度保守的具布鲁菌属特异性的BSP31基因设计了一对引物及探针,并采用矩阵法优化反应体系,筛选出引物、探针、dNTP和Mg2+最优浓度配比,同时进行了特异性和灵敏性试验,建立了实时荧光定量PCR检测布鲁菌的方法。该方法可用于对布鲁菌病普查监测及进出境动物及其产品的检验检疫。     

食源性沙门氏菌的PCR检测

该试验根据GenBank数据库沙门氏菌的特有基因invA(AE008832)设计扩增长度为285 bp的引物,研究PCR技术在检测冷冻肉中沙门氏菌的检测限度.在生理盐水中食源性沙门氏菌的检测限度为103 cfu/mL;在人为污染肉汤中,普通FCR的检出限为1.98×103 cfu/mL;人为污染后的样品肉汤中沙门氏菌浓度为100,101,102 cfu/mL时,分别培养7.6.6h即可检测到.     

应用PCR技术检测鸭疫里默氏杆菌的研究

根据已发表的鸭疫里默氏杆菌15型CVL110／89株编码42－kDa主要外膜蛋白的基因序列设计并合成一对引物，建立PCR方法，对7个血清型的鸭疫里默氏杆菌纯培养菌及野外病死鸭病科组织进行检测。结果表明，7个血清型的鸭疫里默氏杆菌纯培养菌DNA都可扩增出809bp的DNA片段，而对照的2株大肠杆菌和1株沙门氏菌纯培养物DNA扩增结果为阴性；在对24只不同鸭场病死鸭肝、脑的检测中，脑的检出率为19／24（高于细菌分离的13／24），肝脏的检出率为11／24（高于细菌分离的8／24）。由此可见，建立的PCR技术可用于鸭疫里默氏杆菌的鉴定和快速诊断（取脑组织）。但此方法是否对其他血清型的鸭疫里默氏杆菌也适用，有待于收集这些血清型的菌株进行验证。     

鸡源空肠弯曲菌和结肠弯曲菌的临床分离及多重PCR鉴定

弯曲菌(Campylobacter)尤其是空肠弯曲菌(C.jejuni)是重要的人畜共患病病原菌,在人可引起急性胃肠炎,感染严重者伴有心内膜炎、肺炎、败血症、血栓静脉炎、脑膜炎、多发性关节炎以及格林-巴利综合征等疾病[1-3].家禽、家畜、宠物等动物是弯曲菌的常见宿主,因此研究动物源弯曲菌具有重要的公共卫生意义.     

Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell’s serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n?=?5), aborted fetuses (n?=?13), and vaginal swabs (n?=?12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.