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Primer group for gonococci detection, kit containing primer group and application thereof

A detection kit, gonococcus technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of long time, cumbersome procedures, low accuracy, etc.

Active Publication Date: 2014-11-05
THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems of cumbersome procedures, long time-consuming and low accuracy in the existing gonorrhea detection method

Method used

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  • Primer group for gonococci detection, kit containing primer group and application thereof
  • Primer group for gonococci detection, kit containing primer group and application thereof
  • Primer group for gonococci detection, kit containing primer group and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Design of LAMP primer set for detection of Neisseria gonorrhoeae

[0069] The design steps are as follows:

[0070] ①According to Chinese and foreign literature, the genes commonly used in the detection of gonorrhea nucleic acid amplification include 16SrRNA, cppB, orf1, opa, and porA. Among them, porA has shown good sensitivity, specificity, and conservation in clinical applications. In this study, porA was selected. As a target gene for the detection of gonorrhoeae

[0071] ② Search all the porA genes of different strains of Neisseria gonorrhoeae in NCBI for comparison, and mark the mutation-prone sites and mutation frequencies

[0072] ③Select the most conserved porA 600-900bp region as the target sequence for primer design

[0073] ④ Use the online design software Primer Explorer version4 to design specific primers.

[0074] The primer composition and corresponding regions of the LAMP of the present invention are as follows: figure 1 As shown, the sequences of t...

Embodiment 2

[0085] Screening of LAMP primer set and establishment of reaction system

[0086] In order to determine whether the primer set in Example 1 is available, it needs to be screened, and the specific screening steps are as follows:

[0087] 1) DNA extraction

[0088] According to the instructions of Tiangen Biochemical Technology Co., Ltd. Bacterial Genome Extraction Kit, cells were lysed with proteinase K, RNA was removed with RNase A, DNA was adsorbed to the binding column, and DNA was eluted with TE.

[0089] Take 5 μL of DNA solution and add ddH2O to dilute to 1 mL, and use a nucleic acid protein analyzer or an ultraviolet spectrophotometer to measure the optical density at 260 nm and 280 nm. The concentration of DNA was calculated according to the following formula:

[0090] C=A×N×50 / 1000

[0091] In the formula:

[0092] C——DNA concentration (μg / μL)

[0093] A——absorbance value at 260nm

[0094] N——Nucleic acid dilution multiple

[0095] 1OD260nm=50μg / mL double-strand...

Embodiment 3

[0112] Primer set optimization

[0113] A loop primer was introduced into the porA2' primer set to obtain a new primer set porA2 primer set, the sequence of which is shown in the table below.

[0114] Table 1-4 Neisseria gonorrhoeae primer set porA2

[0115]

[0116] Further test the primer set porA2 according to the DNA extraction method, reaction system and LAMP reaction conditions in Example 2, wherein the concentration of each primer in the primer set porA2 in the reaction system is: each of the outer primers F3 and B3 is 0.2 μmol / L , each 1.6 μmol / L of described internal primer FIP and BIP, each 0.8 μmol / L of described loop primer LF and LB, all the other reagents are the same as embodiment 2, and detection result is as follows image 3 shown. Depend on image 3 It can be seen that after the porA2' primer was introduced into the loop primer, the peak eluting time was shortened to 10 min, which greatly shortened the reaction time.

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Abstract

The invention discloses a primer group for gonococci detection. The primer group is designed on the basis of a loop-mediated isothermal amplification technology by using a gonococci PorA gene as a target gene, the primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP, and the nucleotide sequences of the primers are respectively shown in SEQ ID: 2-5. The primer group disclosed by the invention is high in sensitivity and strong in specificity, and can meet the requirement for quick and accurate detection of gonococci. The invention further discloses a gonococci detection kit. The kit can be used for quickly detecting gonococci and has the advantages of short detection time, low cost, high accuracy and the like.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a primer set for detection of Neisseria gonorrhoeae, a kit containing the primer set and applications thereof. Background technique [0002] Sexually transmitted diseases spread quickly and cause great harm. Among them, gonorrhea is widely prevalent in the world, ranking first in the incidence of sexually transmitted diseases all the year round. Its pathogen is Neisseria gonorrhoeae (abbreviated as Neisseria gonorrhoeae). Humans are the only natural host of Neisseria gonorrhoeae, which usually live in the columnar epithelial cells on the mucosal surface, and its main pathogenic substances include pili, outer membrane protein, protease, lipopolysaccharide, etc., and directly infect the genitourinary tract mainly through sexual contact , oropharyngeal and anorectal mucosa, or infection of newborns through the birth canal during delivery, the clinical manifestations ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/04C12Q1/68C12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 郭旭光夏勇江镜全陈琼刘美玲唐晓华
Owner THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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