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Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof

A kit and re-detection technology, applied in fluorescence/phosphorescence, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of no primer pairs and probe tips, no re-detection, and high homology

Active Publication Date: 2014-04-02
苏州华益美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional real-time PCR detection, such as Chinese patents CN1768151A, CN101189505A, CN101273262A, CN101302473A, etc., is only used to detect a single target nucleic acid; Chinese patent CN101624629A discloses (in a single PCR reaction vessel) a real-time PCR method for multiple detection of target nucleic acids in a sample, Execution speed is faster and more integrated, but it is only used to detect three target nucleic acids such as HBV, HCV and HIV, without the practice of more multiplex detection, such as no hints on how to design primer pairs and probes that do not interfere with each other, There is no prompt to adjust the probe concentration
[0006] However, for more complex real-time PCR detection, especially for the five species of Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), and Mycoplasma genitalium (MG) pathogens (among which the three mycoplasmas have high homology), the five-fold non-interfering Primer pairs and probes, designed to cause mutual interference and distort the fluorescence detection curve

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  • Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof

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Effect test

Embodiment 1

[0089] The extraction of embodiment 1 nucleic acid

[0090] Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH) and Mycoplasma genitalium (MG) can be purchased from the Clinical Inspection Center of the Ministry of Health under license conditions, all of which are standard Products for nucleic acid extraction.

[0091] The extraction of nucleic acid is carried out according to the conventional magnetic bead extraction method. In order to adapt to the nucleic acid extraction of the above five microorganisms at the same time, the improvement is as follows: add 90uL nucleic acid extraction solution to 1uL standard (the formula and final concentration are: guanidine isothiocyanate 1.2M, Sodium edetate (pH8.0) 10mM, Tween-20 2% (W / W), sodium perchlorate 1M, ethanol 40% (V / V), Tris-HCl (pH8.0) 10mM) , incubate at 42°C for 10min, then add 10uL D-Beads DNA magnetic bead suspension (50mg / mL, can be purchased from Beijing Aibig...

Embodiment 2 5

[0093] Embodiment 2 Five-fold real-time PCR test

[0094] 1. Primer and probe sequences

[0095] Commission the synthesis of the following primer pairs and probes:

[0096] Primer pairs for detection of Neisseria gonorrhoeae:

[0097] NG-F:ACCGTAACGTCTCTAAGTC

[0098] NG-R: GCAAGATTTCCGATTTGGC

[0099] Probes for the detection of Neisseria gonorrhoeae:

[0100] NG-P: CAGACATCACGCACCGAAGCAT

[0101] Primer pairs for detection of Chlamydia trachomatis:

[0102] CT-F: TGTCCATATCTTTGATACGAT

[0103] CT-R:CTCTGATATTTTGAAGACTCTACA

[0104] Probes to detect Chlamydia trachomatis:

[0105] CT-P:CCAAGCCGAGTCTACAGTTATAGGTCA

[0106] Primer pairs for detection of Ureaplasma urealyticum:

[0107] UU-F: CTGCTCGTGAAGTATTACA

[0108] UU-R:GTACCATCTGGGAAAGTAC

[0109] Probes for the detection of Ureaplasma urealyticum:

[0110] UU-P:ACCAACCATTGTATCTACACCTTCCATA

[0111] Primer pairs for detection of Mycoplasma hominis:

[0112] MH-F:TGTGGTTTAATTTGAAGATACAT

[0113] MH-R:AGTCCT...

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Abstract

The invention relates to a real-time PCR (polymerase chain reaction) method for quintuply detecting target nucleic acid in a nucleic acid extracting solution in a single PCR reaction vessel, which is used for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium in a sample.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection. Specifically, the invention relates to a multiplex real-time PCR method capable of simultaneously detecting five pathogenic pathogens such as Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium, It's fast, ultra-sensitive and done in one step. In addition, the present invention also relates to the reagents involved in the above PCR method, such as primers, probes, etc. that are irrelevant to each other, as well as the detection kit used in the above method and the preparation and application of the corresponding detection kit. Background technique [0002] Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), and Mycoplasma genitalium (MG) are the five main pathogens that cause genitourinary system infections, which can cause urethritis, Prostatitis, cervicitis, s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 陈悦科张文艳刘明霞叶成果王奕江沈靖
Owner 苏州华益美生物科技有限公司
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