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。     

Rapid and sensitive detection of African horse sickness virus by real-time PCR

A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID₅₀ per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of field equine samples. The same primer pair was used to standardise a conventional RT-PCR as an affordable, useful and simple alternative method in laboratories without access to real-time PCR instruments. The two techniques present novel tools to improve the molecular diagnosis of African horse sickness (AHS).     

牛分枝杆菌特异性多重PCR反应在临床样本检测中的应用

本试验应用建立的三重PCR反应检测256份血液样本,结果其中的2份样本呈阳性.应用在线blast进行同源性比较,结果发现,牛分枝杆菌特异性基因IS6110,IS1081和recA与牛分枝杆菌AF2122/97株的同源性均达到99％以上.     

PCR技术在沙门氏菌快速检测中的应用

PCR是一种体外核酸扩增技术 ,具有敏感性强、特异性高、快速、简便等优点 ,在医学、生物学等领域中得到了广泛应用。文章系统地阐述了PCR技术在沙门氏菌快速检测中的应用 ,并对引物基因设计、PCR扩增过程中的技术参数和常见疑难问题做了归纳总结。同时简要介绍了PCR技术的研究进展 ,展望了今后微生物检测的发展方向     

贝类中副溶血弧菌和沙门氏菌荧光定量PCR快速检测方法的建立

为适应进口鲜活水产品的快速检验,有必要建立快速检测多种病原生物的方法。本文针对鲜活水产品中常见的副溶血弧菌和沙门氏菌,设计了特异的荧光引物,建立了检测贝类中这两种细菌的荧光定量PCR体系,并进行了特异性与重复性试验。结果表明,在相同的反应条件下,副溶血弧菌和沙门氏菌均得到了特异性扩增,而阴性对照菌株均未见扩增。副溶血弧菌标准曲线在1.53×105~1.53×109拷贝/μL之间、沙门氏菌标准曲线在4.89×106~4.89×1010拷贝/μL之间有较好的线性关系。与传统的检测方法相比较,该荧光定量PCR方法检测贝类中的副溶血弧菌和沙门氏菌更为快速准确,结果直观,可以满足口岸进口水产品快速检测的需要。     

Detection of Salmonella in faecal, tissue, and feed samples by conventional culture methods and VIDAS Salmonella Test

The VIDAS Salmonella Test (VST) is an enzyme-linked fluorescent immunoassay for the detection of Salmonella-antigens. The suitability of VST for the detection of Salmonella in faecal, tissue, and feed samples was evaluated by the comparison with routine culture methods. From 312 naturally contaminated samples 17 were classified as Salmonella positive by routine methods and 28 by VST. Salmonella were isolated from 15 VST positive samples by the routine method and from eight samples only by an extended culture method. Five positive VST results could not be proved by culture. Two samples were classified as positive by the routine method and as false-negative by VST. The sensitivity varied between 88% and 100% and the specifity between 92% and 100%, depending on the kind of sample. Matrix or serovar specific factors resulting in a false VST result could not be determined. The performance of VST was easy and did not require special experiences. Mostly, samples with Salmonella negative results were faster detected than by culture methods. VST is suitable for the detection of Salmonella in the studied kind of samples especially as a screening method.     

Pouched rats' (Cricetomys gambianus) detection of Salmonella in horse feces

A total of 5 giant African pouched rats detected 10- (95.3% sensitivity) and 100-fold dilutions (78.8% sensitivity) of broth from cultured Salmonella enterica serovar Saint Paul inoculated into dried horse feces. All rats demonstrated some generalization to more dilute concentrations and analyzed 50 samples in less than 20 minutes. These findings suggest that further research examining the use of pouched rats to detect Salmonella is merited.     

鸡粪中沙门氏菌和大肠杆菌O78多重PCR检测方法的建立

为建立同时快速检测沙门氏菌和大肠杆菌O78的方法,本研究根据沙门氏菌inv A基因和大肠杆菌O78 O-抗原特异基因序列设计引物,并在细菌16S r DNA区设计内参引物,通过各项参数调整优化,建立了能够直接从样品中同时检测沙门氏菌和大肠杆菌O78的多重PCR检测方法。结果显示:该方法可以同时扩增出O781 113 bp和沙门氏菌821 bp的特异片段以及细菌16S r DNA 527 bp的通用片段,而对于其他13种肠道致病菌只能扩增出527 bp的细菌同源片段,具有较强的特异性。该体系检测沙门氏菌和大肠杆菌O78的最低检出限分别为10~3cfu/m L和10~2 cfu/m L。增菌5 h,鸡粪模拟污染菌样品中沙门氏菌和大肠杆菌O78的检测灵敏度分别为10~3 cfu/m L和10~2 cfu/m L。本研究建立的检测方法能够在8 h内完成鸡粪样品中两种靶标菌的快速检测,对于沙门氏菌和大肠杆菌O78的鉴别诊断和快速检测具有重大意义。     

Real—time PCR和PCR方法快速检测犬细小病毒

为适应出入境口岸对进出境宠物快速检疫的需要,本研究在建立PCR方法检测犬细小病毒(CPV)的基础上,进一步采用Taqman探针技术建立了快速检测CPV的Real-time PCR方法.通过灵敏度对比试验,证实Real-time PCR方法比PCR方法检测灵敏度显著提高.通过对大量不同采样部位样品的检测证实,本研究建立的Real-time PCR和PCR方法具有较高的可靠性,并可显著提高CPV的阳性检出率.     

The prevalence and PCR detection of Salmonella contamination in raw poultry

Contaminated poultry meat has been identified as one of the principal foodborne sources of Salmonella. The development of rapid detection assays for Salmonella would enable official agencies and food industries to identify contaminated foodstuffs in a more timely manner. In addition, these diagnostic tools could allow more 'real time' decisions to be made regarding end product acceptability. In this study, a survey was carried out to determine the prevalence of Salmonella in raw broiler carcasses. A total of 198 neck skin samples were obtained from within 40 flocks at a commercial broiler slaughtering facility. The presence of Salmonella was assessed by traditional culture methods and by a Salmonella-specific polymerase chain reaction (PCR) test. Salmonella was recovered from 32 (16%) of all samples using traditional culture methods. In contrast, the PCR assay proved to be more sensitive and detected Salmonella DNA in 38 (19%) of the samples tested. The pathogen was detected in 45 (23%) of the 198 samples when culture and PCR results were combined. The sensitivity of the PCR test was also greater than culture when detecting Salmonella from within flocks (53% of flocks by PCR, 30% of flocks by culture). The combination of both tests revealed that 55% of the flocks were contaminated with Salmonella. The PCR assay proved to be a highly specific and sensitive method for detecting Salmonella and the incorporation of a routine PCR test in conjunction with standard culture could be effective in providing a more accurate profile of the prevalence of this pathogen in broiler carcasses.     

饲料中沙门氏菌的PCR检测方法研究

研究应用PCR技术建立饲料中沙门氏菌的快速检测方法,根椐沙门氏菌的invA基因核苷酸序列设计1对特异性引物,分别对4种沙门氏菌的标准菌株及2种非沙门氏菌株进行PCR扩增。结果:4种沙门氏菌均扩增出331bp的特异条带,非沙门氏菌皆无特异条带,特异性达100%,敏感性达104cfu/ml。DNA序列分析证实,所扩增片段为沙门氏菌的invA基因的特异性片断。本研究建立的沙门氏菌检验方法具有较高的特异性和灵敏性,invA基因的序列分析表明,其基因序列较保守,可以做为PCR检验的目的模板。     

Salmonella (sero)types and their resistance patterns in pig faecal and post-mortem samples.

The purpose of this survey was to take stock of porcine Salmonella isolates derived from faecal and post-mortem samples over a 4-year period. Salmonella was isolated by direct inoculation on BGA(NO)-plates (faeces, intestinal content) or sheep blood agar (organs). Antimicrobial susceptibility was tested by the agar diffusion method. Salmonella was isolated in 4.2% of all porcine submissions received at the Animal Health Service. A total of 1305 salmonellae were isolated from a total of 1279 submissions from 1008 different herds. Salmonella Typhimurium was the most frequently isolated serotype (88%), and Salmonella Typhimurium DT104 was the most frequently isolated phagetype (17.2% of Salmonella Typhimurium). Resistance to antimicrobials occurred in 47.3% of all isolates, mainly those of the multiresistant phagetype DT104. Other pathogens were isolated in more than 50% of the submissions. In cases of clinical diarrhoea, multiple pathogens may be involved and therapy and preventive measures should be adjusted accordingly.     

沙门菌PCR快速检测试剂盒的研制与应用

为建立一种简单、快速、灵敏、准确的沙门菌检测方法，根据沙门菌fimY保守基因序列设计合成了1对引物，建立了快速检测沙门菌的PCR方法，并对反应条件进行优化，组装成快速检测试剂盒。结果显示，所有沙门菌均扩增出526bp的特异性条带，而非沙门菌均未扩增出任何条带。检测灵敏度达93CFU，还可检出含3CFU的样品用BP预增菌的菌液或用MM增菌后的菌液，而且稳定性良好，冷冻至少能保存12个月。采用直接菌体加入法、热裂解法、反复冻融法、CTAB碱裂解法和柱式试剂盒提取法分别提取核酸模板，结果CTAB法效果最好，但操作烦琐，而直接菌体加入法和热裂解法可以达到和柱式试剂盒提取法相同的效果，且操作简便、快速、经济、效果好。通过对临床样品的检测，证实该试剂盒具有操作简便、快速、灵敏度高、特异性强、重复性好、稳定性高等优点，值得推广应用。     

Rapid detection of Corynebacterium pseudotuberculosis in clinical samples from sheep

Corynebacterium pseudotuberculosis, a Gram-positive bacterium is the causative agent of caseous lymphadenitis (CLA), a chronic disease of sheep, goats and other warm blooded animals. In the present study, a total of 1,080 sheep reared under semi-intensive system on organized farms situated in the semi arid tropical region of Rajasthan, India, was clinically examined. Pus samples from superficial lymph nodes of 25 (2.31 %) adult sheep showing clinical lesions similar to CLA were collected for laboratory analyses. On the basis of morphological, cultural and biochemical characteristics 12 (48 %) bacterial isolates from pus identified it as C. pseudotuberculosis. A polymerase chain reaction (PCR) assay targeting Putative oligopeptide/dipeptide ABC transporter, nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase coenzyme F420-dependent and proline iminopeptidase (PIP) genes of C. pseudotuberculosis was developed that showed 14 pus samples as positive. All C. pseudotuberculosis isolates were also found positive for these genes in the PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products that showed 98–100 % homology with published sequences available in the NCBI database. The present study shows the incidence of CLA as 2.31 %, 1.1 % and 1.29 % based on clinical, bacterial culture and direct pus PCR assay, respectively. The PCR assay was rapid, specific and as significant as bacterial culture in detecting bacteria directly in the clinical pus samples. The PCR assay developed in the study can be applied for the diagnosis and control of CLA. Furthermore, the assay can also be applied to detect C. pseudotuberculosis in various clinical samples.     

Evaluation of conventional PCR for detection of Strongylus vulgaris on horse farms

Strongyle parasites are ubiquitous in grazing horses. Of these, the bloodworm Strongylus vulgaris is regarded as most pathogenic. Increasing levels of anthelmintic resistance in strongyle parasites has led to recommendations of decreased treatment intensities, and there is now a pronounced need for reliable tools for detection of parasite burdens in general and S. vulgaris in particular. The only method currently available for diagnosing S. vulgaris in practice is the larval culture, which is laborious and time-consuming, so veterinary practitioners most often pool samples from several horses together in one culture to save time. Recently, molecular tools have been developed to detect S. vulgaris in faecal samples. The aim of this study was to compare the performance of a conventional polymerase chain reaction (PCR) assay with the traditional larval culture and furthermore test the performance of pooled versus individual PCR for farm screening purposes. Faecal samples were obtained from 331 horses on 18 different farms. Farm size ranged from 6 to 56 horses, and horses aged between 2 months and 31 years. Larval cultures and PCR were performed individually on all horses. In addition, PCR was performed on 66 faecal pools consisting of 3-5 horses each. Species-specific PCR primers previously developed were used for the PCR. PCR and larval culture detected S. vulgaris in 12.1 and 4.5% of individual horses, respectively. On the farm level, eight farms tested positive with the larval culture, while 13 and 11 farms were positive with the individual and pooled PCRs, respectively. The individual PCR method was statistically superior to the larval culture, while no statistical difference could be detected between pooled and individual PCR for farm screening. In conclusion, pooled PCR appears to be a useful tool for farm screening for S. vulgaris.     

应用PCR技术快速鉴定熊蜂微孢子虫Nosema bombi

基于熊蜂微孢子虫16SrRNA基因序列设计了1对引物,并对引物退火温度和引物浓度等反应条件进行了优化。对所建立方法的敏感性、特异性和稳定性进行了验证后,使用该方法对40份样品进行检测。结果显示,本研究建立了一种能够特异、敏感鉴定熊蜂微孢子虫的PCR方法,对熊蜂微孢子虫总DNA的敏感性达到2.86×10-5 mg/L,可以将熊蜂微孢子虫从西方蜜蜂微孢子虫和东方蜜蜂微孢子等其他可以感染熊蜂的寄生虫中区分出来,具有良好的特异性和稳定性。通过对40份熊蜂样品进行检测结果表明,整个检测过程可以在4h内完成并具有良好的适用性。本研究建立的熊蜂微孢子虫检测方法可用于进境熊蜂的检验检疫。