牛乳中结核分支杆菌复合群的实时荧光PCR检测技术

本研究建立了一种实时荧光PCR检测奶牛乳液中结核分支杆菌复合群的技术，克服了以往牛结核PCR检测样品取样难的缺陷，具有检测灵敏度高、特异性强等优点，可用于奶牛乳房结核的早期诊断以及乳品安全生产检测。以乳液中加卡介苗作为模拟临床检测样本，对DNA提取方法进行改进，利用基于TaqMan技术的实时荧光（real—time）PCR检测。选择分支杆菌编码16sRNA基因和23sRNA基因之间ITS（internal transcribed spacer）序列作为扩增靶序列，研究结果表明卡介苗DNA提取效率提高，实时荧光PCR检测灵敏度达到每毫升乳液检测10个结核分支杆菌。奶牛乳液中结核分支杆菌复合群的检测对兽医系统疫病监测及诊断部门、奶牛养殖业、乳品生产行业有重要应用价值。     

绵羊血液中布氏杆菌DNA提取方法的比较研究

本研究旨在比较5种DNA提取方法对绵羊血液中布氏杆菌DNA的提取效果及对PCR检测的影响。将不同浓度的疫苗株布氏杆菌加入绵羊全血中,采用3种DNA提取试剂盒和酚/氯仿法以及碘化钠法等5种方法提取模拟的绵羊血液样品中的DNA,评价所获得DNA的浓度、纯度和完整性,并采用布氏杆菌特异性PCR进行检测。同时,对各提取方法所需时间及经济成本进行了比较。结果表明,各方法均能提取获得绵羊全血中布氏杆菌DNA,3种试剂盒和碘化钠法获取布氏杆菌DNA的效果相同,而酚/氯仿法获取布氏杆菌DNA的效率最低或存在PCR抑制剂而不适合用于绵羊血液中布氏杆菌的PCR检测。碘化钠法具有耗时较短、成本低、方便的优点,是从绵羊血液中提取布氏杆菌DNA的良好方法。本研究结果为临床绵羊血液中布氏杆菌DNA提取方法的选择提供了参考。     

3种分支杆菌多重PCR快速检测方法的建立

本试验旨在构建一种以不同分支杆菌特异片段为目的基因的多重PCR检测方法。采用GenBank公布的结核分支杆菌RD10序列、牛分支杆菌moaB3序列、鸟分支杆菌16-23SrDNA序列设计并合成特异性引物,扩增产物条带分别为954bp、297bp、119bp。结果显示,三段目的基因都有很高的特异性,对比菌株均无扩增产物出现。对倍比稀释的模板质粒进行检测,该方法的最低检出量为103 copies/μL的DNA模板,该方法特异、敏感、重复性好。首次应用的moaB3基因所得扩增效果良好,成功区分出牛分支杆菌。此多重PCR方法可用于结核分支杆菌、牛分支杆菌、鸟分支杆菌的鉴别和牛结核病的快速临床检测。     

微波加热快速提取芽孢杆菌与乳酸菌DNA

试验旨在研究快速、简单、高效提取芽孢杆菌与乳酸菌DNA的方法。在微波炉额定功率800 W下,对芽孢杆菌和乳酸菌进行微波处理40~150 s,离心获取上清,收获细菌DNA,将样本DNA PCR扩增后进行琼脂糖凝胶电泳验证,测定其纯度和产量后测序。结果显示,在微波炉功率800 W条件下加热40~150 s均可有效提取芽孢杆菌与乳酸菌DNA。所得DNA质量较好（D_{260 nm}/D_{280 nm}在1.8~2.1之间）。DNA浓度符合PCR检测要求,目的条带清晰,所得测序结果满足常规分子生物学研究。该微波提取方法具有简单、快速、高效的特点,且具有广泛的实用性,为乳酸菌与芽孢杆菌快速分子检测提供了简便手段。     

两种动物组织DNA提取方法的比较

本试验以猪耳组织为试验样本,采用酚-氯仿抽提方法[1]和DNA提取试剂盒方法进行DNA提取的对比试验,并通过核酸蛋白定量仪测量了DNA浓度和纯度,并进行了PCR扩增实验。试验表明,两种提取方法都可以进行PCR扩增试验,但采用试剂盒提取方法提取DNA可以直接进行PCR扩增,而酚-氯仿提取法需放置一段时间后效果好,并且采用试剂盒提取的DNA纯度高、速度快、节省耗材、结果稳定、重复性强,适合大量DNA提取试验。     

丝状支原体簇和多杀性巴氏杆菌双重PCR方法的建立及应用

本研究旨在建立丝状支原体簇和多杀性巴氏杆菌的双重PCR检测方法,从而为临床上同时检测这2类病原的感染提供一种更方便、快捷、准确的工具。本研究采用2对特异性检测丝状支原体簇和多杀性巴氏杆菌的引物,对PCR反应体系和反应条件进行了优化,并对双重PCR的特异性及敏感性进行了评价,随后采用该方法对52份临床样本进行了检测。结果显示,所建立的双重PCR方法能同时扩增丝状支原体簇成员和多杀性巴氏杆菌的DNA,而对来源于其他常见病原的DNA均无扩增;对丝状支原体簇和多杀性巴氏杆菌的最低检测限分别为24.8和28.9 pg;能成功地从临床样本中检测丝状支原体簇成员和多杀性巴氏杆菌。结果表明,本研究所建立的双重PCR方法具有很好的特异性和敏感性,为临床丝状支原体簇和多杀性巴氏杆菌感染的快速诊断、病原鉴定及流行病学调查提供了有效的方法。     

3种病原菌多重IMS-荧光RPA检测体系的建立及初步应用

建立一种同时检测沙门氏菌、大肠杆菌O157:H7和布氏杆菌的快速、灵敏、高通量的检测方法。利用特异性免疫磁珠,在37℃条件下从200 mL样液体系中循环捕获目标致病菌。磁珠液提取DNA后,对3种病原菌进行多重IMS-荧光RPA检测。结果表明:针对沙门氏菌、大肠杆菌O157:H7和布氏杆菌的检测限分别达到3.0、4.5和8.7 CFU/mL。使用新建多重IMS-荧光RPA方法对人工感染60份皮张、毛皮纺织品DNA样本进行扩增,结果显示与预期结果一致;对60份公共场所收集的皮张、毛皮、纺织品DNA样本进行扩增,结果显示有2个样本沙门氏菌阳性、1个样本大肠杆菌O157:H7阳性,3份阳性样品送测序,测序结果与GenBank检索沙门氏菌、大肠杆菌O157:H7 DNA序列一致。得出结论:建立的多重IMS-荧光RPA检测体系灵敏度、特异性、准确度符合要求,能够在2 h内完成对3种病原菌检测,可以作为快速应对此三类病原菌安全突发事件的检测手段。     

FQ-PCR方法检测腹泻仔猪肠道内乳酸杆菌和双歧杆菌试验

为了建立一种更有效的动物肠道微生态检测方法,试验设计了对各血清型乳酸菌和双歧杆菌引物序列进行测序。分3批次采集样本,每批分别采集健康和腹泻仔猪新鲜粪样20份。提取每个粪便样本的总DNA。并对解冻后的粪便样本进行荧光定量PCR分析发现,与健康仔猪相比,腹泻仔猪粪便样品中的乳酸杆菌和双歧杆菌mRNA数量有所减少,说明腹泻仔猪肠道中益生菌数量下降,腹泻影响仔猪肠道内环境的健康和微生态平衡。     

牛分支杆菌广西分离株Hsp 65基因的克隆及序列分析

利用牛分支杆菌Hsp 65基因特异性引物,对2株牛分支杆菌广西分离菌株进行PCR扩增,产物经纯化后与载体pMD-18连接,然后转染大肠埃希菌DH5α。提取转染后大肠埃希菌的质粒进行双酶切和PCR鉴定,鉴定为阳性的质粒进行序列测定。测序后通过序列分析软件DNA Star MegAlign对Hsp 65基因核苷酸序列及推导的氨基酸序列进行分析,并与GenBank上已发表的牛分支杆菌的Hsp 65基因进行比较。结果显示,广西分离株mt359、mt370与已发表的7株参考株序列,其核苷酸序列同源性在98.7%～100%之间,推导的氨基酸序列同源性在98.4%～100%之间。表明广西分离的菌株与参考的其他牛分支杆菌菌株的Hsp 65基因差异不大,说明牛分支杆菌分泌蛋白Hsp 65基因十分保守,从而为检测跟踪菌株的变异,研制牛分支杆菌亚单位基因疫苗奠定基础。     

3株猪源非结核分支杆菌16S rRNA基因序列分析

用PCR方法从3株猪源非结核分支杆菌中扩增16 S rRNA基因5′端片段并进行序列测定。用DNA分析软件将所获得的序列与GenBank中分支杆菌序列相比较,计算种间相似性,构建系统进化树,对菌株进行分类与鉴定。结果表明,3株猪源非结核分支杆菌中浅黄分支杆菌1株,偶发分支杆菌2株。非结核分支杆菌在猪中存在的现象应引起重视,对人畜的影响应进一步研究,以便采取有效的防控措施,保护人类及畜群的健康。     

A simplified protocol for molecular identification of Eimeria species in field samples

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.     

Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues.

Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.     

Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis

The high sensitivity of PCR compared to the difficulties of fecal culture in sheep prompted the development of PCR protocols for detection of Mycobacterium avium subsp. paratuberculosis DNA in sheep feces. Although the PCR itself is well developed, and does not pose large technical problems, concentrating the bacteria from samples that may contain low numbers of bacilli using practical methods is still the main difficulty for the use of this technique. In this study, we describe an extraction protocol for the concentration and purification of M. avium subsp. paratuberculosis DNA from fecal samples and we compare it with other methods. The diagnostic performance of the freeze-boiling method was evaluated using a reference method [Vet. Rec. 134 (1994) 95] on fecal samples from a group of selected sheep from different flocks of known individual serological, pathological, and cultural paratuberculosis status. Using, as a reference, a combination of results in those conventional methods, the freeze-boiling PCR protocol showed a sensitivity of 94.1%, and a specificity of 92.3%.     

Determination of an efficient and reliable method for DNA extraction from ticks

Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.     

Evaluation of polymerase chain reaction diagnosis of Cryptosporidium spp in dairy cattle and wildlife

This study investigated the utility of the polymerase chain reaction (PCR) protocol as a screening test for Cryptosporidium spp in 125 fecal samples from dairy cattle and wild rodents. Samples initially examined by fecal flotation and ELISA were evaluated using four PCR protocols (18S SSU rRNA, TRAP-C2, HSP70, and COWP), and the relative accuracy and agreement of PCR protocols was assessed. Although PCR can be both highly sensitive and accurate, the ability of these protocols to accurately detect DNA in samples can vary. A combination of techniques may be the best choice for to screen samples for this parasite.     

An optimized high quality male DNA extraction from spermatophores in open thelycum shrimp species

The crucial step of most of the current genetic studies is the extraction of DNA of sufficient quantity and quality. Several genomic DNA isolation methods have been described to successfully obtain male DNA from shrimp species. However, all current protocols require invasive handling methods with males for DNA isolation. Using Aristeus antennatus as a model we tested a reliable non‐invasive differential DNA extraction method to male DNA isolation from spermatophores attached to female thelycum. The present protocol provides high quality and quantity DNA for polymerase chain reaction amplification and male genotyping. This new approach could be useful to experimental shrimp culture to select sires with relevant genetic patterns for selective breeding programs. More importantly, it can be applied to identify the mating pairs and male structure in wild populations of species as A. antennatus, where males are often difficult to capture. Our method could be also valuable for biological studies on other spermatophore‐using species, such as myriapods, arachnids and insects.     

Implementation of next-generation sequencing for virus identification in veterinary diagnostic laboratories

The value of next-generation sequencing (NGS)-based applications for testing purposes in human medicine is widely recognized. Although NGS-based metagenomic screening may be of interest in veterinary medicine, in particular for intensively farmed livestock species such as pigs, there is a lack of protocols tailored to veterinary requirements, likely because of the high diversity of species and samples. Therefore, we developed an NGS-based protocol for use in veterinary virology and present here different applications in porcine medicine. To develop the protocol, each step of sample preparation was optimized using porcine samples spiked with various RNA and DNA viruses. The resulting protocol was tested with clinical samples previously confirmed to be positive for specific viruses by a diagnostic laboratory. Additionally, we validated the protocol in an NGS viral metagenomics ring trial and tested the protocol on viral multiplex reference material (NIBSC, U.K.). We applied our ViroScreen protocol successfully for 1) virus identification, 2) virus characterization, and 3) herd screening. We identified torque teno sus virus and atypical porcine pestivirus in a neurologic case, determined the full-length genome sequence of swine influenza A virus in field samples, and screened pigs using pen floor fecal samples and chewing rope liquid.     

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods

There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.     

Comparison of four methods of extracting DNA from D. gallinae (Acari: Dermanyssidae)

Dermanyssus gallinae is one of the most serious ectoparasites of poultry and it has been implicated as a vector of several major pathogenic diseases. Molecular detection of such pathogens in mites is crucial and therefore, an important step is the extraction of their DNA from mites. So, we compared four DNA extraction protocols from engorged and unfed individual mites: a conventional method using a Cethyl Trimethyl Ammonium Bromide buffer (CTAB), a Chelex resin, a Qiamp DNA extraction kit and a more recent one filter-based technology (FTA). The DNA samples have been tested for their ability to be amplified by an amplification of a D. gallinae 16S rRNA gene region. The best results were obtained using CTAB and Qiagen methods at the same time with unfed and engorged mites (96% and 100% of amplified samples). FTA produced similar results when using unfed mites but not when processing engorged ones (96% and 70%). Finally, the Chelex method was the least efficient in terms of DNA amplification, especially when applied on engorged individuals (50%). The possible inhibitor role of these Chelex extracted DNA was demonstrated by the means of a PCR control on PUC plasmid. No difference was observed with CTAB, Qiamp DNA extraction kit or FTA methods using DNA extracted one year before.     

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

Formalin-fixed paraffin-embedded tissues (FFPET) represent the largest source of archival biological material available for genomic studies. In this work we present an advanced protocol for extraction of high quality DNA from FFPET that can be applied in several molecular studies. Although cat mammary tumours (CMT) are the third most frequent tumour in cats the recovery of significant number of samples for molecular studies are in some way restricted to FFPET samples. We were able to obtain high quality DNA from FFPET of thirty six CMT that were subjected to pre-fixation and fixation processes routinely used in the veterinary hospitals. The quality of DNA obtained was tested by PCR amplification using six sets of primers that amplify single-copy fragments. The DNA fragments obtained were further sequenced. This protocol was able to provide FFPET gDNA that can be amplified and sequenced for larger fragments up to 1182bp.