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1.
副鸡嗜血杆菌16 S rDNA PCR检测方法的建立 总被引：1，自引：0，他引：1
根据副鸡嗜血杆菌的16 S rDNA基因序列设计一对特异性引物XZIC1和XZIC2,对6株副鸡嗜血杆菌进行PCR扩增。结果显示,该对引物对6株副鸡嗜血杆菌均扩增出与预期大小相一致的282bp片段,而对鸡毒支原体、禽巴氏杆菌、鸡传染性支气管炎病毒、鸡新城疫病毒、大肠埃希菌、鸡白痢沙门菌、禽流感病毒(H9)、鸡喉气管炎病毒及葡萄球菌等9种病原体的扩增结果均为阴性。该PCR敏感性结果表明,本方法可以检测到10pg的副鸡嗜血杆菌DNA模板。采用引物XZIC1和XZIC2,对分别用副鸡嗜血杆菌ctcc253、ctcc255、ctcc257、ctcc269株感染SPF鸡的临床病料DNA进行PCR扩增,均可扩增出单一的282bp的片段。 相似文献
2.
利用aroA基因建立鸡传染性鼻炎PCR诊断方法 总被引：3，自引：0，他引：3
利用aroA基因设计了1对引物，分别对10株标准副鸡嗜血杆菌（HPG）菌株、14株分离的HPG菌株进行PCR扩增，结果均得到了与预期大小一致的片段，而对10株非HPG菌株和3株病毒进行扩增则无相应片段产生；该PCR能检测出10个菌细胞。与常规PCR方法相比，aroA-PCR的敏感性更高。 相似文献
3.
副猪嗜血杆菌PCR检测方法的建立与初步应用 总被引：3，自引：0，他引：3
根据副猪嗜血杆菌16 S rRNA基因设计了一对引物,通过最佳条件摸索扩增出大小为821 bp的特异目的基因片段,建立了快速检测副猪嗜血杆茵的PCR方法,该方法最低检出量达10-3 ng,且对大肠埃希茵、金黄色葡萄球菌、传染性胸膜肺炎放线杆菌和巴氏杆茵等均无交叉反应.用该PCR方法从门诊送检的病料中检测出4株副猪嗜血杆菌,并对分离株SH0854P的PCR扩增产物进行测序与对比分析,其与已发表的GenBank中的相关菌株的同源性为97.3%～100%. 相似文献
4.
为了解副猪嗜血杆菌病在贵州对猪群的危害和流行规律、进一步开展副猪嗜血杆菌病的防治研究提供参考依据,特进行副猪嗜血杆菌PCR检测方法的建立与初步应用研究。根据副猪嗜血杆菌（HPS）16S rRNA基因序列设计1对引物,通过优化组合试验,对贵州省分离的HPS进行PCR扩增,扩增出长为650bp的目的基因片段,而大肠埃希氏杆菌、胸膜肺炎放线杆菌、猪链球菌、葡萄球菌等扩增结果为阴性。采用该方法对贵州发病猪群中350头疑似病例进行PCR检测,检出率为8％～49％。该方法检测副猪嗜血杆菌敏感性高、特异性好,可用于副猪嗜血杆菌的快速诊断。 相似文献
5.
应用环介导等温扩增(LAMP)技术建立了快速特异的检测副鸡禽杆菌的方法。利用在线引物设计软件Primer Explorer Version 4.0,针对副鸡禽杆菌16SRNA基因序列设计两组LAMP引物,并对副鸡禽杆菌特异性DNA的扩增条件进行优化,评价所建立LAMP的特异性和灵敏性。结果显示,优化后的反应条件为65℃恒温反应60min,内引物1.60pmol/μL、外引物0.20pmol/μL。该LAMP对13株副鸡禽杆菌均发生了扩增反应,而对大肠埃希菌、禽多杀性巴氏杆菌、空肠弯曲杆菌等14株其他病原微生物没有扩增反应;该LAMP最低检出量为0.17pg/反应,敏感性远高于常规PCR的56pg/反应。结果表明,所建立的副鸡禽杆菌LAMP具有快速、高效、方便操作等特点,适用于基层兽医部门进行副鸡禽杆菌的快速检测。 相似文献
6.
副猪嗜血杆菌PCR快速诊断方法的建立 总被引：7，自引：0，他引：7
副猪嗜血杆菌营养要求比较苛刻,常规生化试验检测方法比较烦琐,本研究针对副猪嗜血杆菌16SrRNA基因特异性PCR引物序列,合成一对PCR引物,建立相应的PCR检测方法,同时对该方法的灵敏性、特异性等实验。结果显示可检测出浓度为2．8×10^3CFU／mL的副猪嗜血杆菌表明该方法灵敏度高;对大肠杆菌、链球菌、巴氏杆菌、沙门氏杆菌、金黄色葡萄球菌进行PCR扩增均不获得任何条带,表明该方法特异性较强。所以该方法对于临床快速检测副猪嗜血杆菌具有重要意义。 相似文献
7.
产毒素多杀性巴氏杆菌菌落双重PCR检测方法的建立 总被引：1，自引：0，他引：1
为建立快速特异的PCR方法以及同时检测并区分产毒素与非产毒素多杀性巴氏杆菌,本研究根据GenBank登录的多杀性巴氏杆菌KMT1基因和toxA毒素基因序列,设计合成了2对特异引物。特异性试验表明产毒素多杀性巴氏杆菌C51-6扩增出了460bp和1854bp的2条目的片段,而不产毒素多杀性巴氏杆菌、大肠埃希菌、胸膜肺炎放线杆菌、猪链球菌、支气管败血波氏杆菌、副猪嗜血杆菌和鸡白痢沙门菌的扩增均为阴性;敏感性试验表明该PCR方法能从含450CFU的菌液中扩增出相应的目的片段。同时用豚鼠皮肤坏死试验和小鼠致死试验对该PCR方法进行了验证。 相似文献
8.
9.
为建立一种禽多杀性巴氏杆菌的PCR检测方法,本研究根据GenBank中已发表的禽多杀性巴氏杆菌ptfa基因序列,设计与合成了一对特异性引物,建立了一种基于禽多杀性巴氏杆菌ptfa基因的PCR检测方法,并优化了反应条件,检测了该方法的特异性和敏感性。结果显示,该方法可成功扩增出禽多杀性巴氏杆菌ptfa基因片段,而鸡致病性大肠杆菌、鸡金黄色葡萄球菌和鸡白痢沙门菌均未扩增出相应片段。敏感性试验表明该方法可检测最低浓度为1pg/μL的禽多杀性巴氏杆菌基因组DNA。 相似文献
10.
为建立鸭源鸡杆菌(G.anatis)双重PCR检测方法,并对鸡杆菌分离株进行种类鉴定,本研究根据GenBank中G.anatis12656-12株的gtxA基因序列设计一对PCR引物,合成扩增rpoB基因的引物作为内参基因,建立了G.anatis双重PCR检测方法.检测结果显示:以G.anatis Yu-PDS-RZ-1-SLG株DNA为模板进行的双重PCR检测能够扩增出两条目的片段;其它16株细菌包括副鸡禽杆菌、多杀性巴氏杆菌、大肠杆菌、沙门氏菌、福氏志贺菌和奇异变形杆菌均只扩增出了一条目的片段;该方法可以检测到浓度为6.5×102 cfu/mL的G.anatis;34株鸡杆菌分离株均扩增出了两条预期大小的目的片段.此外,序列分析结果表明,10株鸡杆菌河南分离株的rpoB基因序列与鸭源鸡杆菌种参考株(CCUG15563)的同源性最高(97.5％～99.0％),属于鸭源鸡杆菌种.本研究建立的G.anatis双重PCR检测方法,可用于含gtxA基因鸡杆菌菌株的鉴定及临床病原学诊断. 相似文献
11.
J K Miflin X Chen R R Bragg J M Welgemoed J M Greyling R F Horner P J Blackall 《The Onderstepoort journal of veterinary research》1999,66(1):55-57
Seventy five bacteria tentatively identified as Haemophilus paragallinarum (the causative agent of infectious coryza), eight identified as Ornithobacterium rhinotracheale and 13 identified as NAD-independent Pasteurella species were isolated from chickens with respiratory infection in various provinces in South Africa. The isolates were characterized by conventional biochemical and serological methods. A polymerase chain reaction (PCR) assay specific for H. paragallinarum was used to identify the cultures directly from colonies. The PCR assay gave positive results for all isolates that were identified by conventional methods as H. paragallinarum, irrespective of whether they were nicotinamide adenine dinucleotide (NAD)-dependent (43 isolates) or NAD-independent (32 isolates). The eight isolates that were identified by conventional methods as O. rhinotracheale and the 13 isolates identified as various Pasteurella species gave negative results in the PCR assay. This study has demonstrated that colony PCR is a rapid method for uniquely identifying both NAD-dependent and NAD-independent strains of H. paragallinarum and distinguishing them from other bacteria, such as O. rhinotracheale and Pasteurella species. 相似文献
12.
Strains of Bisgaard taxon 31, isolated from chickens in South Africa suffering from a respiratory disease with clinical symptoms and gross lesions similar to infectious coryza, showed great phenotypical similarities with Haemophilus paragallinarum infection except for NAD requirement, beta-galactosidase activity and maltose fermentation. Deoxyribonucleic acid-deoxyribonucleic acid hybridization confirmed a high level of genetic relatedness (DNA binding value, 89%) with Haemophilus paragallinarum. Guanine + cytosine content and genome size data also support the classification of taxon 31 strains within the species Haemophilus paragallinarum. 相似文献
13.
Soriano VE Longinos GM Fernández RP Velásquez QE Ciprián CA Salazar-García F Blackall PJ 《Avian diseases》2004,48(4):886-889
The virulence of the reference strains of the nine currently recognized Kume serovars of Haemophilus paragallinarum was investigated. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. All nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). Our results indicate that virulence differences exist among the serovars of H. paragallinarum. 相似文献
14.
A total of 60 isolates of Haemophilus spp. from chickens, including four reference strains of H. paragallinarum and one of H. avium, were examined for their physiological and biochemical properties. The isolates could be placed into two groups. One group was identified as H. paragallinarum and consisted of 43 isolates including the four reference strains of H. paragallinarum. The other group was identified as H. avium and consisted of 17 isolates including the reference strain of H. avium. H. avium can be differentiated from H. paragallinarum by its possession of the enzymes catalase and alpha-glucosidase, capacity to grow in air, production of acid from galactose, and by the fact that its growth is not improved by the addition of chicken serum. In addition, the majority of H. avium isolates, unlike H. paragallinarum, possess a yellow pigment and produce acid from trehalose. 相似文献
15.
Strains of the bacterium Ornithobacterium rhinotracheale (ORT), a causal agent of respiratory diseases in birds, were microbiologically isolated, identified, and molecularly characterized. Blood-enriched culture media and biochemistry tests were used for microbiologic identification. Polymerase chain reaction (PCR) and repetitive extragenic palindromic PCR (rep-PCR) techniques were used for molecular identification and characterization, respectively, of the microorganism. ORT strains were isolated in enriched media from the trachea and air sacs of broilers, breeders, and layers from several geographic zones of Peru. Of the original 75 strains isolated from 75 clinical samples from which ORT was recovered during 1998-2000, 25 were selected for further study based on ORT as the primary pathogenic isolate (no other pathogens were detected). Selected isolates were molecularly identified and characterized by PCR using specific primers designed from the conserved zones of the 16S ribosomal genes. Primers used for the identification of ORT produced a specific fragment of 784 base pair (bp), which did not appear in Haemophilus paragallinarum or Pasteurella multocida, microorganisms with similar morphologic and biochemical characteristics that produce dinical signs identical to those of ORT. All 25 strains of ORT tested with rep-PCR had a genetic profile similar to that of ORT American Type Culture Collection 51463, indicating the presence of only one genotype in the ORT strains studied. 相似文献
16.
Development of a multiplex PCR and PCR-RFLP method for serotyping of Avibacterium paragallinarum 总被引：1，自引：0，他引：1
Sakamoto R Kino Y Sakaguchi M 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2012,74(2):271-273
Avibacterium (Haemophilus) paragallinarum (A. paragallinarum) is a causative agent of infectious coryza in chickens and is classified into three serovars by agglutination tests. In an effort to identify the serovars easily, PCR and PCR-RFLP were employed. As the target gene for PCR, the hypervariable region of HMTp210, which encodes the HA antigen, was used. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively. Alternatively, the 1.6 kbp fragments were amplified with another primer pair encompassing the hypervariable region and was subjected to digestion with Bgl II, which resulted in the detection of serovar-specific digestion patterns. These results indicate that PCR and PCR-RFLP using the hypervariable region of HMTp210 are alternative methods to identify the serovar of A. paragallinarum. 相似文献
17.
Thirty-nine Australian isolates of Haemophilus paragallinarum were compared serologically with 3 reference serotype strains of H. paragallinarum using a plate agglutination test. Twenty-eight of the isolates were serotype C, 5 were serotype A, while the remaining 6 isolates could not be assigned to a serotype. 相似文献
18.
Soriano VE Téllez G Hargis BM Newberry L Salgado-Miranda C Vázquez JC 《Avian diseases》2004,48(4):890-895
The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used for fingerprinting of reference strains and Mexican isolates of Haemophilus paragallinarum. A total of nine ERIC patterns were given by the nine serovar reference strains of this bacteria. Two Modesto (C-2) reference strains from different sources showed the same ERIC pattern. Seventeen ERIC patterns were obtained among 29 Mexican isolates included in the study, belonging to serovars prevalent in Mexico (A-1, A-2, B-1, and C-2). Obtained results indicate that the ERIC-PCR technique could be used as a molecular laboratory tool for subtyping of H. paragallinarum. 相似文献
19.
副鸡嗜血杆菌的分离鉴定及我国鸡传染性鼻炎的流行状况分析 总被引：8，自引：0，他引：8
从不同地区免疫失败鸡场的鸡传染性鼻炎疑似鸡体内共分离到19株细菌，经回归试验、PCR等方法鉴定为副鸡嗜血杆菌。又将分离株用血清平板凝集试验、血凝抑制试验和型特异性单抗进行了血清型的鉴定，结果表明分离菌有15株为A型，4株为B型，说明我国鸡传染性鼻炎的流行已经出现了新的特点，在疾病预防和控制中应加以注意。另外大多数鸡场的免疫失败是由于免疫效果不良所致，但也有相当一部分鸡场的免疫失败是由于B血清型的出现引起的。 相似文献