Reagent kit for rapidly and synchronously detecting Chlamydi trachomatis, Urealpasma parvum and Ureaplasma urealyticum

Abstract

The invention discloses a reagent kit for quickly and synchronously detecting chlamydi trachomatis, fine urealyticum and ureaplasma urealyticum, more particular relates to a reagent kit for detecting the genes of a sexually transmitted disease pathogen. The reagent kit mainly comprises DNA nucleic acid extracting solution, a positive plasmid containing the detection sequence of three pathogens and a TaqMan PCR amplification system jointly detected by a single tube and single enzyme by adopting the one-step method, wherein the three pathogens comprise the chlamydi trachomatis, the fine urealyticum and the ureaplasma urealyticum. The invention has the advantages of good specificity, high sensitivity, accurate quantification, quick detection speed, simple use step and high repeatability, can simultaneously detect samples with high flux, and not only can carry out synchronous quantitative detection to the chlamydi trachomatis, the fine urealyticum and the ureaplasma urealyticum, but also can replace the traditional culture method and ELISA (enzyme linked immunosorbent assay) diagnostic method used always.

Description

technical field

[0001] The invention relates to a sexually transmitted disease pathogen gene detection kit, in particular to a kit for synchronous detection of Chlamydia trachomatis, Ureaplasma parvum and Ureaplasma urealyticum, which has the characteristics of high sensitivity, strong specificity and rapidity. Background technique

[0002] Ureaplasma urealyticum (UU for short) and Chlamydiatrachomatis (CT for short) are common sexually transmitted disease pathogens, and they are often co-infected in patients with clinical nongonococcal urethritis (NGU). Among sexually transmitted diseases, Ureaplasma urealyticum is related to non-gonococcal urethritis and epididymitis; it is obviously related to prostatitis, female urethral symptoms, pyelonephritis, and pelvic inflammatory disease, or suggests an etiological role.

[0003] In recent years, researches on the pathogenic mechanism and epidemiology of Ureaplasma urealyticum have received sufficient attention. Recent studies ha...

Examples

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...

technical field

[0001] The invention relates to a sexually transmitted disease pathogen gene detection kit, in particular to a kit for synchronous detection of Chlamydia trachomatis, Ureaplasma parvum and Ureaplasma urealyticum, which has the characteristics of high sensitivity, strong specificity and rapidity. Background technique

[0002] Ureaplasma urealyticum (UU for short) and Chlamydiatrachomatis (CT for short) are common sexually transmitted disease pathogens, and they are often co-infected in patients with clinical nongonococcal urethritis (NGU). Among sexually transmitted diseases, Ureaplasma urealyticum is related to non-gonococcal urethritis and epididymitis; it is obviously related to prostatitis, female urethral symptoms, pyelonephritis, and pelvic inflammatory disease, or suggests an etiological role.

[0003] In recent years, researches on the pathogenic mechanism and epidemiology of Ureaplasma urealyticum have received sufficient attention. Recent studies ha...

Examples

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...

Images