Nucleic acid probe combination, kit and method for detecting common pathogens in genital tracts

A technology of nucleic acid probes and pathogens, applied in the field of biological diagnosis and detection, can solve the problems of inability to detect multiple target genes or pathogens, high requirements for nucleic acid quality and quantity, and low sensitivity of target sequences, and achieve visualization of detection results and strong specificity , high detection sensitivity and accuracy

Pending Publication Date: 2021-06-29
SICHUAN HUAHAN TRIO BIOTECH CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, the sensitivity of this method to detect the target sequence in the sample to be tested is not high, the resolution is poor, and false positives and false negatives are prone to occur; and this method has relatively high requirements for the quality and quantity of the detected nucleic acid
At the same time, this method can only perform single-plex reactions. One reaction cell can only detect one gene or pathogen in one reaction, and cannot specifically detect multiple target genes or pathogens. It is not a combination of multiple pairs of primers in the traditional sense. multiplex PCR

Method used

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  • Nucleic acid probe combination, kit and method for detecting common pathogens in genital tracts
  • Nucleic acid probe combination, kit and method for detecting common pathogens in genital tracts
  • Nucleic acid probe combination, kit and method for detecting common pathogens in genital tracts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 is used to detect the design and preparation of primers and probes for reproductive tract pathogens

[0053] 1. Multiplex PCR assay design

[0054] Multiplex PCR (multiplex PCR), also known as multiple primer PCR or composite PCR, refers to a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments. The main difficulty is to ensure the specific amplification of primers and avoid the interaction between different primers in the same system.

[0055] (1) Sequence analysis and primer design

[0056] Query the whole genome sequence of each pathogen at NCBI, and after determining the highly conserved genes, design the specific detection primers for each pathogen in the specific sequence segment of each pathogen, including upstream primers and downstream primers; PCR amplification is used for the designed primers Carry out preliminary detection of amplification efficiency...

Embodiment 2

[0110] Embodiment 2 is used for the preparation of the membrane chip kit that detects genital tract pathogen

[0111]In this example, a membrane chip kit for detecting pathogens in the reproductive tract, including multiplex PCR reaction solution, sample extraction solution, positive control substance, negative control substance, instructions, membrane chip, hybridization tube, hybridization reaction solution, box body, etc. The membrane chip consists of 7 groups of probe sequences (SEQ ID No.3, 6, 9, 12, 15, 18, 21), a group of positive control probe sequences (SEQ ID No.24) and A set of endogenous control probe sequences (SEQ ID No.25), the 5' end of each probe is labeled with amino group (NH 2 ), the upstream primer or downstream primer was labeled with biotin.

[0112] Wherein, the multiplex PCR reaction solution contains 8 pairs of upstream and downstream primers of Example 1, PCR buffer, MgCl 2 , dUTP, dNTPs, C-PC, Taq enzyme and UNG enzyme;

[0113] MgCl 2 The conce...

Embodiment 3

[0116] The detection method of embodiment 3 reproductive tract pathogen nucleic acid detection kit (membrane chip method)

[0117] 1. Utilize the kit in Example 2 to detect Mycoplasma hominis / Neisserial gonorrhoeae / Chlamydia trachomatis / Ureaplasma urealyticum / Mycoplasma genitalium / Human cytomegalovirus / Herpes simplex virus type II in human genital tract secretions, specifically Proceed as follows:

[0118] (1) Nucleic acid extraction: Take 10 swabs of female genital tract secretions, wash them fully in 1 mL of normal saline, squeeze dry the swabs and discard, transfer 500 μL of the liquid to a 1.5 mL centrifuge tube, centrifuge at 13,000 rpm for 5 min, discard For the supernatant, add 50 μL of the sample extract to the precipitate, vortex and mix well, then bathe in 56°C for 10 minutes, then bath in 95°C for 2 minutes, centrifuge at 13,000 rpm for 5 minutes, and take the supernatant for PCR reaction;

[0119] (2) Multiplex PCR amplification: Take 5 μL of the nucleic acid extr...

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Abstract

The invention discloses a nucleic acid probe combination, a kit and a method for detecting common pathogens in genital tracts. The nucleic acid probe combination comprises one or more groups of multiplex specific primer combinations and / or one or more probe combinations sequentially fixed on the surface of a membrane chip. The primer combination comprises primers of common pathogens in genital tracts and primers of endogenous control GAPDH genes. The probe combination comprises a specific probe for common pathogens in genital tracts, a positive dot hybridization probe and an endogenous control GAPDH gene probe. According to the invention, a multiplex PCR and reverse dot hybridization combined technology is adopted to solve problems of low assay accuracy of conventional primers and probes on pathogens in genital tracts and difficulty in realizing parallel assay of single reaction systems for more than seven pathogens in genital tracts. The nucleic acid probe combination of the present invention overcomes defects of easiness in false positive result caused by cross contamination and low accuracy in multiplex reaction systems in the prior art, and has the advantages of quick detection, convenience in operation, high accuracy and sensitivity, good specificity and visible detection results.

Description

technical field [0001] The invention belongs to the technical field of biological diagnosis and detection. More specifically, it relates to a nucleic acid probe combination, kit and method for rapid detection of common pathogens in the reproductive tract; specifically Mycoplasma hominis / Neisserial gonorrhoeae / Chlamydia trachomatis / Ureaplasma urealyticum / Mycoplasma genitalium / Human giant cell Virus / herpes simplex virus type Ⅱ identification methods, primers, probes and kits. Background technique [0002] Reproductive tract infection (Reproductive Tract Infection, RTI) is a common disease and frequently-occurring disease in women. The general term, including vaginitis, vulvitis, pelvic inflammatory disease and other infections. Female reproductive tract infection is a global social and public health problem, characterized by high incidence, high recurrence rate, and wide prevalence. Reproductive tract infection is the source of many diseases. If it is not diagnosed and trea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/35C12R1/36C12R1/01C12R1/93
CPCC12Q1/705C12Q1/689C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/101C12Q2521/531
Inventor 黄盈李文静郎小亮周航黄广平
Owner SICHUAN HUAHAN TRIO BIOTECH CO LTD
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