Reagent kit for rapidly and synchronously detecting Chlamydi trachomatis, Urealpasma parvum and Ureaplasma urealyticum
A technology for Chlamydia trachomatis and Ureaplasma urealyticum, which is applied in biochemical equipment and methods, and microbial measurement/inspection, and can solve problems such as poor specificity, harm to experimenters, and false positive results
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Embodiment 1
[0060] Embodiment 1: kit composition and preparation
[0061] a. DNA extraction reagent (lysate)
[0062] Prepare for yourself, lysate: 50mmol / L NaOH, 10mmol / L Tris-HCl, pH8.0, volume fraction 1% Triton X-100, volume fraction 1% NP-40, 0.5mmol / L EDTA pH8.0 .
[0063] b. Composition of fluorescent PCR 10×Buffer:
[0064] 500mM KCl, 100mM Tris-HCl (PH9.025°C), 1.0% Triton X-100;
[0065] c. Fluorescent quantitative PCR reaction solution: PCR 10×buffer 5.0μl, 10μmol / L UP forward primer and reverse primer 2.5μl each, 10μmol / L UU forward primer and reverse primer 2.5μl each, 10μmol / LCT forward Primer and reverse primer 2.0 μl each, UP 5 μmol / L fluorescent probe 2.5 μl, UU 5 μmol / L fluorescent probe 2.5 μl, CT 5 μmol / L fluorescent probe 2.0 μl, 25 mmol / L MgCl 2 3μl, 10mmol / L dNTPs 1.0μl, 5U / μl HOTSTART Taq DNA polymerase 1.0μl, and then supplement the volume with double distilled water to make the total volume of the reaction solution 50μl.
[0066] d. Standard positive templa...
Embodiment 2
[0068] Implementation Example 2: Simultaneous detection of Chlamydia trachomatis, U. parvum and Ureaplasma urealyticum using kits
[0069] a. Add 1ml of sterile normal saline to the specimen test tube, shake well, transfer to a 1.5ml centrifuge tube, centrifuge at 10000g for 5min, and repeat washing once. Add 50 μl of DNA extraction solution directly to the precipitate, mix thoroughly, bathe in boiling water for 10 minutes, centrifuge at 10,000 g for 5 minutes, and take 2 μl of the supernatant for PCR reaction.
[0070] b. Serially dilute the positive standard template (reagent d) to 10 8 copies / μl, 10 7 copies / μl, 10 6 copies / μl, 10 5 copies / μl, 10 4 copies / μl, 10 3 copies / μl, 10 2 copies / μl, 10 1 copies / μl, 10 0 copies / μl.
[0071] c. Take 18 μl of fluorescent quantitative PCR reaction solution (reagent c) respectively, take 1 μl of UP DNA obtained in step a) and 1 μl of UP positive standard template diluted in step b), and take UU DNA obtained in step a) and the UP...
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