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A compound PCR kit for detecting hps, ss2, pm, app
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A kit and upstream technology, applied in multiplex PCR detection and biological fields, can solve problems such as time-consuming, difficult diagnosis, low sensitivity, etc., and achieve the effect of saving time
Active Publication Date: 2021-10-12
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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These diseases are mostly mixed infection or secondary to other pathogenic infection, causing serious economic losses to the pig industry
[0003] The clinical symptoms of Haemophilus parasuis, Streptococcus suis, Pasteurellosis multocida, and porcine infectious pleuropneumonia are similar, and multiple infections and provocative infections make diagnosis more difficult
At the same time, it has the disadvantages of long time consumption and low sensitivity
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Embodiment 1
[0031] Embodiment 1 HPS, SS2, Pm, APP compound PCR reaction system and optimization of conditions
[0032] Using rTaq enzyme, dNTPs, total primer volume and Mg 2+ The combination of four factors and four levels of orthogonal experiment, the results are shown in Table 1.
[0033] Table 1 Optimization of HPS, SS2, Pm, APP compound PCR reaction system
[0034] (Four factors and four levels of orthogonal test)
[0038] Example 2, determination of optimal reaction system and conditions for HPS, SS2, Pm, APP composite PCR
[0039] Then optimize the multiplex PCR annealing temperature, cycle number, extension time, etc., and finally determine the PCR reaction system and conditions, see Table 2.
[0040] Table 2 The optimal reaction system for multiplex PCR
[0041]
[0042] The PCR amplification conditions were: 94°C for 5min, 94°C for 30s, 58°C for 30s, 72°C for 50s, 72°C for 10min, 4°C for 2h. 30 Cycles include 94°C for 30s, 58°C for 30s, and 72°C for 50s.
Embodiment 3
[0043] Sensitivity test of embodiment 3, HPS, SS2, Pm, APP composite PCR
[0044] Using the optimal reaction system and conditions of multiplex PCR, the amplification of the sensitivity test was carried out, and the results are shown in figure 2 . from figure 2 It can be seen that the lowest detection amount of HPS is 112.5pg / μL, the lowest detection amount of SS2 is 11.7pg / μL, the lowest detection amount of Pm is 8.7ng / μL, and the lowest detection amount of APP is 267pg / μL.
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Abstract
The invention belongs to the multiple PCR detection in the field of biotechnology, in particular to a composite PCR kit for detecting HPS, SS2, Pm, APP, which can detect Haemophilus parasuis (HPS), Streptococcus suis (SS2), Toxigenic Pasteurella multocida (Pm) and Actinobacillus pleuropneumoniae (APP), the invention solves the problem of mixed infection of HPS, SS2, Pm and APP, and the clinical symptoms are difficult to distinguish and treat PRDC problems, and fast, high accuracy, saving detection time and cost.
Description
technical field [0001] The invention belongs to the field of multiplex PCR detection in the field of biotechnology, and specifically relates to a specific detection method for Haemophilus parasuis (HPS), Streptococcus suis type 2 (SS2), Pasteurella multocida (Pm), porcine toxigenous Actinobacillus pleuropneumoniae (APP) composite PCR rapid detection kit and its application method. Background technique [0002] Respiratory diseases have always been a major problem plaguing my country's pig industry, among which Haemophilus parasuis, Streptococcus suis, toxin-producing Pasteurella multocida, and porcine infectious pleuropneumonia are the most common respiratory diseases in pigs. The most important bacterial infectious disease. Haemophilus parasuis can cause fibrinous pneumonia, arthritis and meningitis in pigs. Streptococcus suis can cause purulent meningitis, septicemia, endocarditis, pneumonia and arthritis, etc. Among them, type 2 streptococcal suis is an important zoonoti...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 袁芳艳田永祥刘泽文刘威周丹娜杨克礼段正赢郭锐高婷
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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