Absolute fluorescent quantitative PCR primer pair for detecting Lawsonia intracellularis, kit and detection method thereof
A technology of fluorescence quantification and Lawsonia, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc. It can solve the problems of inability to truly reflect the infection of pigs, low sensitivity, and inability to quantify bacteria, etc. problems, to achieve the effect of convenient primer design, avoid cross-contamination, and improve viability
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Embodiment 1
[0048] 1. DNA extraction of Lawsonia intracellulare from porcine ileum tissue samples:
[0049] (1) Take about 25mg of porcine ileum samples frozen at -80°C, put them in a 2ml centrifuge tube, and cut them into pieces as much as possible with scissors
[0050] (2) Add 180 μL of Buffer LS-2, 20 μL of Proteinase K and 10 μL of RNase A (10 mg / ml), and warm in a 56°C water bath until the tissue is completely lysed. (It takes about 2 to 3 hours, and the sample can be taken out from time to time for shaking or pipetting to speed up the lysis)
[0051] (3) Centrifuge at 12000rpm for 2 minutes to remove impurities.
[0052] (4) Add 200 μL Buffer BS-2 and 200 μL 100% ethanol to the lysate, and mix well by pipetting.
[0053] (5) Transfer the above solution to a Universal DNA Mini Column, let stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min at room temperature, and discard the filtrate.
[0054] (6) Add 500 μL of Buffer WA to the Mini Column, centrifuge at 12,00...
Embodiment 2
[0077] Example 2 The primary detection application of the absolute fluorescence quantitative PCR detection method in clinical samples.
[0078] The 178 suspected PPE samples collected clinically were detected by IFA and absolute fluorescent quantitative PCR. The results (Table 2) showed that 93 positive samples were detected by IFA in 178 diseased materials, and 93 positive samples were detected by IFA in 3 different regional slaughterhouses. The rates were 54.2%, 57.9% and 42.9% respectively; while 99 positive samples were detected by the qPCR method, the positive detection rates in the slaughterhouse were 56.9%, 61.4% and 47% respectively, indicating that the established detection method can be used in clinical applications .
[0079] Table 2
[0080]
PUM
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