| Actinobacillus pleuropneumoniae(APP) and Haemophilus parasuis(HPS) are major primary pathogens of porcine respiratory disease complex, and Salmonella(Sal) is a secondary pathogen of this disease. The three pathogenic bacteria can cause serious infectious diseases with high mortality and morbidity rates in pigs. Those diseases have created the great economic loss to the pig industry around the world. The early diagnosis is key to combat the diseases. Currently, the available detection methods for the three pathogenic bacteria include conventional testing techniques, DNA microarray and PCR assay. But these assays have some drawbacks of time consuming, low sensitivity and cannot meet the requirement of rapid detection in large batches. For guiding diagnosis and treatment in the early phase of the disease, it is necessary to establish high throughput, simple, rapid, specific, sensitive and stable methods for detection of the three pathogenic bacteria: APP, HPS and Sal.In this research, we developed three sandwich DNA hybridization assays(DNAH) and three loop-mediated isothermal amplification assays(LAMP), respectively, to detect APP, HPS and Sal. The new detection technology for early diagnosis and treatment of epidemic disease, disease surveillance and epidemiological investigation provides an effective protection to reduce the harm of infectious diseases in swine production and promote healthy and stable development of livestock. And it is precursory and important to study as a new detection technique reservation.Experimental process and results were as follows:1. The nucleotide sequences of Apx IVA genome of APP strains, 16 S r RNA genome of HPS strains, inv A genome of Sal strains were retrieved from the Gen Bank, and aligned by using the MEGA5.0 multiple sequence alignment program. The capture probe and detector probe of sandwich DNA hybridization assay were designed on the basis of the Apx IVA(or 16 S r RNA, inv A) alignments by using primer 5 software. The six LAMP primers were designed on the basis of the Apx IVA(or 16 S r RNA, inv A) alignments by using an online software(Primer Explorer V4). The feasibility and specificity of all sets of probes and primers were then subsequently validated by BLAST program on NCBI.2. Under the optimized concentration of probes, ratio of hybridization solution and probe solution, hybridization temperature and hybridization time, the DNAH assays for rapid detection of APP, HPS, Sal were established, respectively. The developed methods were carried out for evaluation of its specificity, sensitivity, reproducibility and stability. The results showed that the detection limits of the APP-DNAH assay was 0.46 CFU/m L. No cross-reactivity was observed with other related bacteria. This method was precise, the intra-assay coefficient of variation(CVi) was between 8.30 % and 10.29 %, and the inter-assay coefficient of variation(CVo) was between 16.78 % and 20.07 %. The limit of detection for HPS was 0.31 CFU/m L and no false-positive results were observed for the other related bacteria. The CVi ranged from 10.97 % to 16.22 %, while the CVo ranged from 18.32 % to 22.50 %. Sal-DNAH accurately detected three salmonella reference strains and no specifically cross-reacted with other bacterial pathogens. The lower detection limit was 1.2 CFU/m L. The CVi ranged from 16.53 % to 17.97 % and the CVo ranged from 16.33 % to 22.10 %.3. Optimization of the reaction conditions included reaction temperature, and inner and loop primer concentrations, the LAMP assays for rapid detection of APP, HPS, Sal nucleic acids were established, respectively. These methods were evaluated on its specificity, sensitivity, reproducibility and stability. The results showed that specific amplification was only observed in APP by using APP-LAMP. There was no cross-reactivity found between self-designed primers and other eleven pathogens and the negative control. The limit of detection was 0.307 fg/μL per reaction volume. The assay has the favorable reproducibility and stability, and the CVi was 0.67 % and the CVo was 2.21 %.The detection limit for HPS by the LAMP assay was 0.175 fg/μL. The assay did not cross-react with other eleven pathogens. The CVi was 0.76 % and the CVo was 3.84 %. The detection limit of Sal-LAMP method was 0.810 fg/μL. The DNA templates of three different salmonella serotypes would be enough to amplify specific positive bands and other ten pathogens had no amplified products. The CVi was 1.47 % and the CVo was 2.45 %.4. Lungs of 204 specimens were analyzed using the developed DNAH, LAMP assays and nested PCR exposed in standard SN/T 1447-2011 to determine presence of infection by APP. The results showed that the positive rates of three kinds of methods were 4.9 % and the accordance rate was 100%. DNAH and LAMP assays in the experiment spend less time as compared with the nested PCR assay.Nasal swab of 195 specimens were analyzed using the developed DNAH, LAMP assays and PCR assay exposed in standard NY/T 2417-2013 to determine presence of infection by HPS. The results showed the positive rate of HPS by DNAH and LAMP were 4.62 %, and detected by PCR was 3.08 % lower than the two methods developed in the study. Besides, the overall process of PCR was time-consuming.Disease pork of 371 specimens were analyzed using the developed DNAH, LAMP assays and PCR assay exposed in standard SN/T 1869-2007 to determine presence of infection by Sal. The results showed that the positive rate of Sal by DNAH and LAMP were 6.2 %, and detected by PCR was 5.7 %. Compared with PCR method, DNAH and LAMP assays have the features as short time, high detection rate.In conclusion, our newly developed DNAH and LAMP detection methods for APP, HPS, Sal, respectively, are more specific, sensitive and stable. Those methods also could achieve rapid and accurate identification for infected specimen of single pathogen in a short span of time(Pathogen detection requires 1.5 hours of DNAH and 1 hour of LAMP). Those methods may serve as effective means for screening bacteria in clinical specimens and controlling disease. |
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