| Bordetella bronchiseptica is a pathogenic bacterium with a broad range of numerous mammals, results in acute and chronic respiration system disease. Human infections occasionally occur, especially the children and immunocompromised individuals.The B. bronchiseptica could increasing incidence and severity of respiratory system disease through cooperate with other pathogenic germs.for example, B. bronchiseptica and Pasteurella multocida are etiologic agents of progressive atrophic rhinitis (PAR) in swine, this disease can cause pig physical growth, reduce feed utilization, cause huge economic losses in animal husbandry. Therefore, it is necessary to developed a rapid and accurate method to detect the B. bronchiseptica for preventing and cure the AR.This study is divided into two parts, as follows:The first part, the study of sequence alignment software by DNAstar flagellum gene (Genbank accession number:EU327790, AJ012319, L13034) on B. bronchiseptica, find out the homologous sequence, design the online primer at the website of LAMP primer design. The 25μL of the LAMP reaction system was carried out after The reaction system and conditions was Optimized. The specificity of the LAMP assay.The result confirmed that the LAMP method was highly specific for B. bronchiseptica, because it showed a ladder pattern with a set of bands of different sizes appeared only using the DNA from the B. bronchiseptica as template. The sensitivity of the LAMP assay. The result showed that the detection limit of LAMP assay being estimated to be 9 copies/tube, PCR reached the sensitivity limit of 9×101 copies/tube, this assay suggested that the sensitivity of the LAMP assay was approximately 10-fold greater than that of the PCR assay. The applicability of the LAMP assay for detecting B. bronchiseptica in clinical samples was performed. The 31 swine lung suspected disease(46 nasal swabs) were detected bacteria, PCR, LAMP three kinds of methods, the positive rate of bacterial separation, PCR method, LAMP method were 9.67%(3/31),19.4%(6/31),32.3%(10/31),while the positive rate of suspected three methods of infection group was 19.57%(9/46),58.14%(20/46), 69.77%(28/46), and the LAMP and bacterial isolation, PCR positive coincidence rate was 100%, the clinical detection of the LAMP method is more suitable for B. bronchiseptica.The second part, from 2012 April to 2013 April, the LAMP method established in this study and 438 samples of nasal swab samples from Sichuan Province were analyzed by Bb and toxigenic Pasteurella using PCR method for detection of toxigenic multocida according to Townsend et al (T+Pm) detection, epidemiological survey was conducted by Bb and T+Pm in large scale pig farms in parts of Sichuan Province, the prevalence of Bb and T+Pm in Sichuan province, According to the Bb survey results, the positive rate of Bb in small scale farms under 30 days of age,30-90 day old,90 day old piglets were above 24.29%(17/70),39.19%(29/74) and 21.79% (17/78),the positive rate of Bb and large-scale farms under 30 days of age,30-90 day age, over 90 days old piglets were 32.31%(21/65),40.26%(31/77),20.27% (15/74),application of agglutination test for detection of serum samples, the results showed 438 serum samples, the total positive rate was 51.59%, Bb antibody positive small scale farms under 30 days of age,30-90 day old,90 day old piglets above rates were 37.14%(26/70),51.35%(38/74),55.13%(43/78),large scale farms were respectively 38.46%(25/65),63.64%(49/77),60.81%(45/74).Combined with the clinical manifestations of Bb and T+Pm negative pigs, show or pathogen carriers of recessive infection has a considerable number of Sichuan Province in herds, if not promptly eliminate hidden infected pigs, will make the outbreak and spread the cost disease, caused great harm to the pig farm. |
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