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.     

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.     

RT-PCR检测猪瘟病毒方法的建立与应用

建立RT PCR检测猪瘟病毒的方法。根据已发表的猪瘟病毒E2基因 (囊膜糖蛋白gP55基因 )序列 ,设计合成了一对特异性引物 ,扩增片段的大小为 50 7bp ,用RT PCR技术对石门系标准株和 1 0株分离株进行检测。结果这对引物对标准株和 1 0株分离株均能扩增出与预期大小相符 50 7bpRT PCR产物 ,而对其他 6种猪病病原核酸的扩增结果为阴性。该RT PCR可检出 1 0 0pg的猪瘟病毒RNA模板 ,对人工感染猪不同组织样品进行检测 ,结果对白细胞抽提的核酸样品检出率最高为 1 0 0 % (2 4 / 2 4 ) ,其次为扁桃体、脾、肾 ,检出率为 83 3 % (2 0 / 2 4 ) ,再者为淋巴结 ,检出率为66 7% (1 6/ 2 4 )。对送检的 1 9份疑似猪瘟的病死猪病料组织进行RT PCR检测 ,结果有 1 6份样品为猪瘟病毒阳性。兔体交叉反应试验结果RT PCR阳性的 1 6份病料中 ,有 1 4份样品被判为含有猪瘟病毒 ,其他病料兔体交叉反应试验结果全为阴性     

RT-PCR技术诊断猪瘟的应用研究

应用反转录—聚合酶链反应(RT-PCR)对猪瘟进行诊断应用研究。应用RT-PCR况对来自广西不同地区的135份疑似猪瘟病料进行检测，以份诊断为阳性，阳性率62．2％。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份，经RT-PCR检测，37份为阳性，阳性率为13．4％。其中健康猪扁桃体带毒较高，246份扁桃体中有35份阳性，占14．2％。采自柳州健康猪的26份淋巴结材料全为阴性，只有邕宁县的1份健猪淋巴结阳性。结果表明，RT-PCR技术可应用于猪瘟的临床诊断。     

RT-PCR检测猪传染性胃肠炎病毒方法的建立

猪传染性胃肠炎 ( TGE)是一种以严重腹泻、呕吐和脱水为临床特征的高度接触性传染病 ,属于国际兽疫局 ( OIE)法典中 B类疫病必检的猪传染病。TGEV是一种有囊膜的正链 RNA冠状病毒。目前检测 TGEV的方法主要有中和试验、免疫荧光抗体试验、酶联免疫吸附试验和电镜技术 ,这些方法对于检测 TGEV和防制 TGE起到了重要作用 ,但也存在特异性不强、灵敏性差、耗时长等缺点。c DNA探针、RT- PCR近年来已成为国际上用于检测病原微生物的主要方法。由于 c DNA探针与临床样品中非相关核酸有一定的非特异性结合 ,尤其是检测粪便、尿液等…     

Use of dried blood smears for detection of feline hemoplasmas using real-time polymerase chain reaction

The objective of the current study was to determine the sensitivity and specificity of real-time polymerase chain reaction (real-time PCR) for feline hemoplasmas when applied to DNA extracted from dried whole-blood smears in comparison to that for DNA extracted from liquid whole blood. Blood samples were collected into ethylenediamine tetra-acetic acid tubes from 305 cats with possible or suspected hemoplasmosis, and dried blood smears from each sample were prepared. DNA was extracted from blood smears and a 160-microl aliquot of each liquid blood sample by using a robotic extractor and was subjected to real-time PCR for feline glyceraldehyde-3-phosphate dehydrogenase (liquid blood), 18S ribosomal RNA (dried blood), and "Candidatus Mycoplasma haemominutum", Mycoplasma haemofelis, and "Candidatus Mycoplasma turicensis" DNA. When using the results for liquid whole blood as the gold standard, the sensitivity of each assay for "Ca. M. haemominutum", M. haemofelis, and "Ca. M. turicensis" was 49 of 66 (74%), 11 of 13 (85%), and 11 of 20 (55%), respectively. The specificity of each assay was 224 of 234 (96%), 287 of 287 (100%), and 280 of 280 (100%), respectively. When possible, liquid blood samples should be submitted for detection of feline hemoplasmas by using real-time PCR. The improved sensitivity of real-time PCR on blood smears for M. haemofelis compared with that of the other hemoplasma species may reflect the higher organism burdens associated with infection with this species.     

A real-time polymerase chain reaction assay for the detection of the myxozoan parasite Henneguya ictaluri in channel catfish

Proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya ictaluri, is the most prevalent parasitic infection affecting commercial channel catfish (Ictalurus punctatus) aquaculture. There are currently no effective chemotherapeutic or biological control measures for PGD, which often peaks during the spring and fall when water temperatures are between 16-25 degrees C. The current diagnostic techniques of gross examination of gill clip wet mounts and histopathology are subject to false-negatives during the early stages of infection, and the quantifiable nature of end-point polymerase chain reaction (PCR) is subjective. Consequently, a rapid and more sensitive quantitative real-time PCR assay was developed for the detection of H. ictaluri during the early stages of infection in channel catfish. A 23 base-pair TaqMan probe was designed based on previously published H. ictaluri PCR protocols. The sensitivity of the assay was the equivalent of a single H. ictaluri actinospore, and in a pond challenge study, quantitative real-time PCR proved to be more sensitive than gross examination, microscopic examination of gill clip wet mounts, and histopathologic examination of gill tissue sections. Future applications of this assay will focus on developing methodologies to be used in conjunction with current pond-monitoring protocols to evaluate potential treatments and better manage this significant seasonal disease.     

RT-PCR检测猪繁殖障碍与呼吸道综合征病毒方法的建立与应用

作者参考国内外已发表猪繁殖障碍与呼吸道综合征病毒(PRRSV)的基因序列和PRRSV相关的RT—PCR检测方法报道，根据PRRSV高度保守、高特异性的NP蛋白基因设计了一对引物，通过对疫苗毒株、细胞分离株的检测，建立了PRRSVRT—PCR检测方法。进一步对40份临床样品进行了检测应用，结果有17份阳性样品，与临床症状和血清学结果相符。     

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi

OBJECTIVE: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. SAMPLE POPULATION: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. PROCEDURE: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. RESULTS: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.     

新型反转录-复合套式聚合酶链式反应(RT-nPCR)鉴别猪瘟强毒和弱毒疫苗方法的建立

对GenBank中登录的25株猪瘟病毒强毒株和兔化弱毒疫苗株基因组全序列进行比较分析,在其高度保守区设计1对通用引物,扩增片段为609bp,并在该对引物扩增区域内设计针对疫苗弱毒的特异引物,扩增片段为237bp,建立一种能够区分猪瘟病毒强毒和疫苗弱毒的敏感、特异、重复性好的反转录-复合套式聚合酶链式反应(RT-nPCR)鉴别诊断方法。该方法能将我国大陆地区流行的不同基因亚群的猪瘟病毒强毒与疫苗弱毒完全区分开来,且不与牛病毒性腹泻病毒及其他猪源病毒发生非特异反应。应用本试验建立的反转录-复合套式聚合酶链式反应(RT-nPCR)方法可以及早对猪瘟作出准确诊断,并可将强毒感染猪迅速从弱毒疫苗免疫猪群中筛选出来,减少了未感染免疫猪被误杀的可能性。