The Developmet Of The LAMP Method For Mycoplasma Hyopneumoniae Diagnosis And Clinical Investigation

Mycoplasma hyopneumoniae(Mhp) is an important pathogenic cause swine enzootic pneumonia. It Was considered an important pathogen to cause porcine respiratory syndrome (PRDC).Mhp is widely popular in China, its main symptoms are coughing, gasping, etc. It also prone to other pathogens such as mycoplasma hyorhinis, Pasteurella multocida. Mycoplasma hyopneumoniae infection, according to the level of virulence of the mycoplasma strains and individual pigs can cause chronic or acute infection.lt can be infected by pigs of different age, sex and varieties, especially in the high susceptibility of piglets and weaned piglets,it can cause higher morbidity and mortality, causing serious economic losses.Mycoplasma hyopneumoniae infection can be controlled through vaccination and drug treatment. Weather to accurately detect the causative pathogen in order to take appropriate measures, is an important way to disease control. Pathogens were isolated and cultured the most basic way to check a pathogen, But the pathogen were isolated and cultured to identify Mycoplasma hyopneumoniae have several shortcomings:First of all,Mycoplasma hyopneumoniae have serious growth conditions, and have the long incubation period, the first train takes about10days in order to grow visible colonies; Another aspect is its growth is often overshadowed by other pathogens such as mycoplasma hyorhinis. So far, the number of molecular diagnostic methods have been studied and applied, in which PCR method is a most widely used and most commonly used laboratory molecular diagnosis. The loop-mediated isothermal amplification (LAMP) is a new and efficient nucleic acid amplification method, Compared with the PCR method, the LAMP is simple, less time-consuming and does not require the use of Laboratory sophisticated instruments and equipment, and only need a bath to complete the entire reaction. In my present study, in order to establish a rapid diagnosis on Mhp.We conserved Mycoplasma hyopneumoniae P36gene as a target gene, the establishment of the PCR method of detection of Mycoplasma hyopneumoniae to LAMP amplification method, its main results are as follows:1.the development of LAMP methodLAMP amplification of the P36gene as a target gene, designed four specific primers, the reaction temperature is62℃,60min reaction, PCR method similarly to the P36gene as a target gene,94℃denaturation, annealing of55℃,68℃extension. The results can be observed by electrophoresis imaging system. Create a PCR method as a verification and evaluation of the LAMP method. The results can be observed by electrophoresis gel imaging system. Through the use of dilution evaluation of the sensitivity of the two methods, the results show the sensitivity of the PCR reaction for0.292ng Mhp DNA and the sensitivity of the LAMP reaction is0.0292ng Mhp of DNA,.In465clinical lung tissue and398nasal swabs detection, the LAMP method more positive than PCR method, especially in the detection of toxic low nasal swab samples, LMAP method detection rate reached19.6%but PCR only6.0%Therefore, the LAMP method has higher sensitivity than the PCR method.Distribution and detecting of clinical samples。2.the clinical use of LAMP method2.1LAMP method for detection of clinical samplesThe LAMP method for the detection of465copies of the submission of clinical lung tissue as well as the acquisition of398nasal swab samples from seven provinces:Hubei, Hunan, Guangxi, Guangdong, Jiangxi, Shanxi, Henan,.Detection and classification of clinical samples by two methods showed that the detection rates in the different provinces have an average of40%-50%. Some provinces such as Hubei Province, and even up to56%, we can conclude that the classification samples of the Fattening Pigs, he detection rate is66.7%, accounted for11.2%of breast-feeding pigs. Therefore, in Mhp the prevention and treatment, to increase the Mhp finishing pigs treated with the control. In over whether the immunity the the Mhp vaccine detection rate of the statistics, we found that the unvaccinated pigs, the detection rate is29.1percent, while the vaccinated pigs the detection rate decreased to13.3%It can be inferred that the Mhp vaccine can reduce the detection rate of the Mhp.2.2LAMP method to detect artificially infected with Mhp detoxification Using artificial trachea infection in the artificial infection of Mycoplasma hyopneumoniae, strains gift of168standard virulent strain of the Jiangsu Provincial Academy of Agricultural Sciences, the amount of attack agents (1×1010CFU/mL)2mL. After7d,14d,21d,28dcollected nasal swabs for detoxification detection. The nasal swab test results show that the herd incidence detected in the detoxification of time is consistent. Observations suggest that swine disease in the18-21d,21d test results can be found the most of the pigs have detoxification,28d results showed that all incidence pigs were detoxification,. It can detect detoxification after artificially infected14d2.3.the detection of Mhp and PCV-2co infectionThe statistics of the co infection of Mycoplasma hyopneumoniae and porcine circovirus type Ⅱ (PCV-2) virus, the same PCR method used for detection of PCV-2. The test results showed that the detected465lung tissue, the co infection of Mhp and PCV-2reached14.0%, while the PCV-2alone infection rate reached26.2%, so in order to control Mhp infection, prevention and control of the PCV-2is also very important.