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

A re-detection and kit technology, applied in the field of nucleic acid detection, can solve problems such as no primer pair and probe prompt, no primer pair and probe design, no prompt probe concentration adjustment, etc.

Active Publication Date: 2013-01-23
苏州华益美生物科技有限公司
View PDF10 Cites 11 Cited by
  • 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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof

Examples

Experimental program
Comparison scheme
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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap