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Detection product for detecting genital tract pathogens by MALDI-TOF MS and application

A pathogen and reproductive tract technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of complex operation and long time consumption, and achieve simple data analysis, high biological value, and specificity Good results

Active Publication Date: 2021-08-06
BIOYONG TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods inevitably have problems such as complicated operation and long time consumption.

Method used

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  • Detection product for detecting genital tract pathogens by MALDI-TOF MS and application
  • Detection product for detecting genital tract pathogens by MALDI-TOF MS and application
  • Detection product for detecting genital tract pathogens by MALDI-TOF MS and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] Embodiment 1, primer design

[0151] All detection target regions in the present invention are single-region detection, and the PCR primers correspond to the 3' ends of the above-mentioned reference PCR primers to ensure amplification. The properties were adjusted without affecting the specificity of the primers and without increasing the non-specificity of the primers.

[0152] Primers were designed for the target detection segments of 10 pathogens (the nuc segment of Staphylococcus aureus, the scpB segment of Group B Streptococcus, etc.), and after searching the NCBI database, the source of the target segment of each pathogen is as follows:

[0153] The target fragment of Staphylococcus aureus: the target fragment of Staphylococcus aureus is selected from the nuc fragment, > DQ399678.1:1-450 Staphylococcus aureus nuclease (nuc) gene, partial cds, and its sequence number is DQ399678.1;

[0154] The target segment of group B streptococcus: the target segment of group B...

Embodiment 2

[0171] Embodiment 2, build the detection mass spectrometry model of 10 kinds of pathogens

[0172] Using ABI 9700 PCR instrument, according to the instructions, the specific sites related to 10 kinds of reproductive tract pathogens were tested.

[0173] In this example, a multiplex primer system for detecting 10 kinds of reproductive tract pathogens was used to construct a mass spectrometry model for the detection of 10 kinds of pathogens. Set the blank control as deionized water, the negative control as human genomic DNA, and the positive control as the plasmid mixture of each target for detecting 10 kinds of reproductive tract pathogens.

[0174] The components used in the kit for PCR, PCR product purification and single base extension are:

[0175] serial number component name main ingredient Packing Specifications 1 Reaction solution I dNTPs, Tris-HCl, MgCl 2

323μL / tube×1 tube 2 Enzyme I Amplification enzyme, UNG enzyme 23μL / tube×1 ...

Embodiment 3

[0206] Embodiment three, isolate patient's pathogenic DNA

[0207] Sample source: vaginal secretions. Sampling personnel should wear clean hands and wear disposable gloves before sampling, and should avoid the menstrual period of the subject when collecting. The specimen collection and extraction methods are as follows: Cervical sampling swab: Rotate a plastic rod swab with a polypropylene fiber head and gently insert it into the vagina, stay for a while and then rotate clockwise 5 times. Take out the swab and dip it into a tube containing 3ml of sampling solution, discard the tail, and tighten the cap of the tube. The swab preservation solution should not be stored for more than one week at 2-8°C, and should not be stored for more than one month at -20°C. It can be transported in a curler with ice or a foam box with ice. It is recommended to use fresh swab preservation solution as much as possible.

[0208] Sample extraction: Bacterial DNA extraction kits were used to extra...

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Abstract

The invention provides a primer group, a product and a non-diagnostic detection method for detecting typing specific sites of 8 genital tract pathogenic bacteria and 2 human papilloma viruses by a time-of-flight mass spectrometry method. The genital tract pathogenic bacteria comprise staphylococcus aureus, group B streptococcus, neisseria gonorrhoeae, gardnerella vaginalis, candida albicans, mycoplasma genitalium, mycoplasma urealyticum, chlamydia trachomatis, HPV16 virus types and HPV18 virus types. A novel method for detecting 10 genital tract pathogens by clinical mass spectrometry combined with multiple PCR is firstly provided, the multiple PCR, single base extension, mass spectrometry detection and other technologies are integrated into a whole, a detection template is amplified through the PCR technology, a trace sample is detected through the mass spectrometry, and the advantages of the two technologies are synthesized. The method is far superior to that for detecting pathogen specific fragments by singly using PCR, and has higher detection sensitivity.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and relates to a detection product and application for quickly identifying 10 kinds of reproductive tract pathogens by using laser time-of-flight mass spectrometry technology. Background technique [0002] According to the latest survey report of the World Health Organization, for adult women, the most common disease is not a cold, but a reproductive tract infection, and the incidence rate is as high as 90%; the most common symptoms are not fever, cough, headache, etc. Vaginal itching, odor, pain, frequent urination, urgency, etc. Moreover, the occurrence of many other diseases in women is also closely related to the abnormal function of the reproductive system, such as osteoporosis, coronary heart disease, diabetes, and chloasma. In a sense, the problem of the reproductive system is related to the health and happiness of a woman's life, and it can also be said to be the "source of all...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6895C12Q1/686C12Q1/14C12Q1/04C12N15/11
CPCC12Q1/708C12Q1/689C12Q1/6895C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2533/101C12Q2565/627C12Q2545/113Y02A50/30
Inventor 李维邬文燕张雪莲张海燕马庆伟
Owner BIOYONG TECH
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