丝状支原体簇及绵羊肺炎支原体双重PCR方法的建立及应用

为了快速检测和鉴别丝状支原体和绵羊肺炎支原体,试验采用2对检测丝状支原体和绵羊肺炎支原体的特异性引物,成功建立了一种能同时检测丝状支原体和绵羊肺炎支原体的双重PCR方法.结果表明:该方法特异性强、敏感性高,对临床病料的检测效果好.     

猪支原体肺炎流行病学和诊断技术研究进展

论文概述了猪支原体肺炎的病原学特性、流行病学和诊断方法的研究进展,并对猪支原体肺炎临床检测、血清学检测、分子生物学检测等检测技术的优缺点进行比较.在猪支原体肺炎预防控制方面,对上述检测技术和诊断方法的应用进行了讨论和展望,旨在为研究该病提供理论参考.     

牛支原体套式PCR检测方法的建立

为建立牛支原体检测方法,本研究采用牛支原体的oppD/F基因的两对特异性引物,建立了牛支原体的套式PCR检测方法.该套式PCR可以从100 ccu/mL(color change unit颜色变化单位)的牛支原体培养物中检出目的片段;同时还可以从病牛肺脏、病牛鼻拭子中扩增出目的片段,而且结果与病原分离结果一致.特异性试验结果表明,该方法与其它支原体无交叉反应,是一种特异性强、敏感性高的牛支原体检测方法.     

支原体PCR检测方法的建立和应用

依据GenBank中登录的常见细胞污染支原体16S rRNA基因序列,选择高度保守的区域设计引物,建立了一种快速灵敏的支原体PCR检测方法.该方法可以特异性检测出6种支原体,并能敏感的检测到10个拷贝数的目的基因.采用建立的PCR方法和传统的培养法对23个不同样品(细胞、血清、疫苗成品、半成品)进行检测,符合度100％.该方法的建立可大大缩短疫苗生产过程中支原体检验所需时间.     

牛支原体、无乳支原体和丝状支原体丝状亚种小克隆三重PCR检测方法的建立及初步应用

本研究旨在建立一种可一次性区分牛支原体、丝状支原体丝状亚种小克隆和无乳支原体的三重PCR诊断方法,为临床诊断和流行病学调查提供可靠检测技术.根据GenBank发表的上述3种病原的基因组序列保守区域设计3对特异性引物建立三重PCR方法;确定其检测敏感性,以猪支原体、鸡支原体、无乳支原体和丝状支原体丝状亚种小克隆基因作模板检验其特异性;同时和病原分离鉴定结果对比其准确性.结果表明在优化体系和条件下能够同时得到扩增长度为448、549、375 bp 3条特异性片段,未扩增出猪、鸡支原体模板特异性片段;其敏感性(可检测到的最小模板DNA含量)为0.8 ng·μL-1;36份临床样品检测结果显示,三重PCR检测结果与分离培养鉴定方法一致,均能鉴定出牛支原体阳性病料.本研究建立的三重PCR诊断方法能够一次性鉴别3种支原体,具有高敏感性、特异性和准确性,可用于临床诊断和流行病学调查.     

牛支原体双重PCR检测方法的建立

为鉴别牛支原体(Mycoplasma bovis)与丝状支原体丝状亚种SC(M.mycoides subsp.mycoides SC),本实验通过优化M.bovis特异性引物pMB81-1/pMB81-2s和MmmSC特异性引物SC1/SC2的退火温度,建立了鉴别M.bovis和MmmSC的双重PCR检测方法。该方法能够分别由M.bovis和MmmSC扩增得到528bp和270bp片段。敏感性试验结果显示该方法检测M.bovis和MmmSC培养物的最低浓度分别为106cfu/mL和105cfu/mL。特异性试验结果显示,该方法对无乳支原体代表株PG2、丝状支原体丝状亚种LC型代表株Y-goat、山羊支原体山羊肺炎亚种Mccp、绵羊支原体Y-98、猪鼻支原体BST-7、巴氏杆菌以及结核分枝杆菌扩增结果均为阴性。应用该方法对临床病料的检测结果与培养鉴定结果的符合率为100%,表明该方法具有良好的特异性和敏感性,可以应用于临床检测。     

Leachii支原体PCR检测方法的建立与应用

为建立一种leachii支原体的检测方法,本研究根据leachii支原体LPPA外膜蛋白编码基因序列设计一对引物,建立了PCR检测方法。结果显示,该方法仅对leachii支原体扩增出539 bp的目的片段,而与牛支原体、丝状支原体山羊亚种、绵羊肺炎支原体、丝状支原体丝状亚种小菌落型、山羊支原体山羊肺炎亚种等其它支原体无交叉反应,具有良好的特异性;该方法对leachii支原体的最低检测浓度为105 ccu/m L,敏感性较高。采用该方法可以在乳汁和关节液等病料样品中检测到leachii支原体,与分离培养的结果相符。上述结果表明,本研究建立的leachii支原体PCR检测方法具有特异性强、敏感性高的特点,可实现准确、高效检测leachii支原体的目的。     

IHA检测SPF猪与非SPF猪肺炎支原体抗体比较

猪肺炎支原体可引起猪喘气病(MPS).猪感染肺炎支原体后约2周,可在其血清中检测到该抗体.本文应用IHA对SPF猪、非SPF猪血清中肺炎支原体抗体进行检测比较,并对此方法进行评价.     

猪鼻支原体TaqMan MGB探针荧光定量PCR方法的建立

为建立快速检测猪鼻支原体(M.hyorhinis)的荧光定量PCR方法,本研究根据GenBank中登录的p37基因序列设计并合成引物及MGB探针,构建含有p37基因的重组质粒,以其为标准品绘制标准曲线,并检测该方法的特异性、敏感性和重复性.结果显示该方法具有良好的特异性,与猪肺炎支原体、猪絮状支原体、猪滑液支原体、鸡毒支原体、副猪嗜血杆菌、猪胸膜肺炎放线杆菌、猪圆环病毒、猪瘟病毒及猪流感病毒无交叉反应,对M.hyorhinis的检测敏感性为10拷贝/μL,并且稳定性好,变异系数小于2％.利用建立的荧光定量PCR方法对55份临床样品进行检测,阳性检出率为87.3％(48/55),而分离培养方法和普通PCR的阳性检出率分别为41.8％(23/55)和29.1％ (16/55).该结果表明,建立的方法特异性强、敏感性高、稳定性好,可以用于M.hyorhinis临床样品的检测,对M.hyorhinis的快速和定量检测具有重要意义.     

山羊和绵羊嗜血支原体PCR检测方法的建立与应用

为建立羊嗜血支原体(M.ovis)病快速检测方法,本实验根据GenBank中登录的羊M.ovis 16S rRNA基因序列设计一对引物,以山羊和绵羊M.ovis基因组DNA为模板,建立M.ovis PCR检测方法,并进行特异性、敏感性及临床应用试验。结果显示,建立的M.ovis PCR检测方法扩增片段大小为508 bp,与GenBank中M.ovis参考株同源性达99%以上,该方法与猪嗜血支原体、牛嗜血支原体等病原体无交叉反应,最低检测40个拷贝的DNA;通过对延边地区60份山羊和绵羊血液样本的检测结果表明,建立的PCR检测方法具有特异、敏感、准确等优点,完全适用于M.ovis的检测。     

兽用疫苗中霉形体污染的套式PCR检测技术建立及初步应用

根据GenBank公布的鸡毒霉形体、猪肺炎霉形体、猪滑液霉形体和絮状霉形体的16s rRNA基因序列,利用引物设计软件DNAStar和Primer5.0自行设计3对特异性引物,以市场上常见的兽用疫苗为检测对象进行试验,优化反应体系,建立了兽用疫苗霉形体污染检测的套式PCR方法。同时,本试验还对市场上的兽用疫苗进行随机抽查检测,其检出率分别为24.2%(23/95)、21.1%(20/95)和14.7%(14/95)。     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

Development of real-time PCR assay for the detection of Mycoplasma bovis

Mycoplasma bovis is one of the important bovine mycoplasma involved in economically important clinical conditions like respiratory diseases, otitis media, and mastitis. The present study was undertaken with the objective of developing a SYBR Green dye-based real-time PCR assay targeting uvrC gene for the diagnosis of M. bovis. The analytical sensitivity and specificity of the assay were evaluated. The test showed 10³-fold more sensitivity than conventional PCR and detected down to 100 fg level of DNA. It was found to be specific, as no cross reactivity was shown with other related bacteria and Mycoplasma species. The developed assay was able to detect down to 40 copies of uvrC gene from spiked bovine milk samples. At present, this developed assay may be used as a valuable diagnostic tool for the detection of Mycoplasma bovis.

     

Design,optimization, and application of a conventional PCR assay with an internal control for detection of ‘Candidatus Mycoplasma turicensis’ 16S rDNA in domestic cats from Brazil

Background: ‘Candidatus Mycoplasma turicensis’ (CMtc) is a hemotrophic bacterial species that can, alone or in combination, induce anemia in cats. The diagnostic test of choice for hemoplasma infections is PCR. Conventional PCR assays have been developed for the detection of Mycoplasma haemofelis (Mhf) and ‘Candidatus M. haemominutum’ (CMhm) but not for CMtc. Although real‐time PCR assays have been reported for all of the feline hemoplasmas, the expense of necessary instrumentation precludes its use in Brazil and many other countries. Objectives: The goals of this study were to develop and optimize a conventional PCR assay to diagnose CMtc using an internal control to detect false‐negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil. Methods: Species‐specific primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10‐fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica‐based protocol and tested using the PCR assay. Results: Primer concentration, annealing temperature, and MgCl₂ concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc. Conclusion: These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the first time the prevalence of CMtc infection in domestic cats in Brazil.     

Diagnosis of feline haemoplasma infection using a real-time PCR assay

Haemobartonella felis has been reclassified within the genus Mycoplasma as Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum', collectively referred to as the feline haemoplasmas. A total of 78 cats from the Johannesburg area that had blood samples submitted to a private veterinary laboratory were tested using a real-time polymerase chain reaction (PCR) assay able to detect and distinguish the two feline haemoplasma (basonym Haemobartonella) species. All samples had been diagnosed with haemoplasma infection by cytological examination of blood smears. Statistical analysis was performed to evaluate associations between haemoplasma status, age, and haematological and biochemical parameters. On PCR assay 43 cats (55%) were haemoplasma negative, 25 (32.1%) positive for 'Candidatus Mycoplasma haemominutum', 5 (6.4%) positive for Mycoplasma haemofelis and 5 (6.4%) positive for both species. Significant inverse correlation was found between the amount of M. haemofelis DNA present in the blood and the haematocrit value. Cats that were positive for M. haemofelis showed macrocytic regenerative anaemia, monocytosis and thrombocytopaenia. This report documents the existence of both haemoplasma species in cats in South Africa.     

The occurrence of the feline “Candidatus Mycoplasma haemominutum” in dog in China confirmed by sequence-based analysis of ribosomal DNA

In the present study, polymerase chain reaction (PCR) assay was used to detect haemoplasmas (haemotropic bacteria) in 40 clinically healthy pet dogs in Foshan city, Guangdong Province, China, and one dog was found positive. Comparison of its 16S ribosomal DNA (rDNA) sequence with relevant sequences showed that the isolated haemoplasma had greater sequence identity to feline species “Candidatus Mycoplasma haemominutum” (99%) than to “Candidatus Mycoplasma haematoparvum” (95%). This result, for the first time, indicates the presence of the feline “Candidatus Mycoplasma haemominutum” in Chinese dogs and it represents the first survey of its kind in China by using PCR assay. The results indicated that dog may represent one of the hosts for the feline “Candidatus Mycoplasma haemominutum”.     

Molecular detection of Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' Infection in cats by direct PCR using whole blood without DNA extraction

Detection of hemotropic Mycoplasma spp. infection was attempted in cats by PCR using whole blood without DNA extraction. A total 46 of 54 (85%) cats with suspected Mycoplasma spp. infection showed a positive reaction, corresponding completely with the results of standard PCR testing. The direct PCR assay was sensitive enough to detect more than 0.0061% parasitemia for ;C. M. haemominutum' and 0.0075% parasitemia for M. haemofelis. These data indicate that the direct PCR assay might be sufficient for use as a tool in clinical examinations.     

A multiplex PCR to identify porcine mycoplasmas present in broth cultures

Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.     

Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay

The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP-based identification procedures of 17 different field isolates agreed with those obtained by conventional methods.     

Detection and Identification of Mycoplasma conjunctivae in Infectious Keratoconjunctivitis by PCR. Based on the16S. rRNA. Gene

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 × 10⁵ by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).