CN110317889B

CN110317889B - Gonococcus nucleic acid detection kit and gonococcus nucleic acid detection method

Info

Publication number: CN110317889B
Application number: CN201910360402.5A
Authority: CN
Inventors: 华鑫; 陈明峰; 高虹
Original assignee: SHENZHEN GOLDSITE DIAGNOSTICS Inc
Current assignee: SHENZHEN GOLDSITE DIAGNOSTICS Inc
Priority date: 2019-04-28
Filing date: 2019-04-28
Publication date: 2023-04-18
Anticipated expiration: 2039-04-28
Also published as: CN110317889A

Abstract

The invention discloses a gonococcus nucleic acid detection kit and a gonococcus nucleic acid detection method, which comprise an upstream primer, a downstream primer and a probe, wherein the sequences are as follows: an upstream primer AAAGCCTTCCCAACTCG; downstream primer CGCCCAAATGGTTACTTC; probe 5'-x-TGGTCGTCTATCCGTTCCGT-y-3'; wherein x is a fluorescence reporter group, and y is a fluorescence quencher group. The kit provided by the invention has the advantages of strong specificity and high sensitivity, the detection method consumes less time, and the gonococcus nucleic acid can be quickly and accurately detected for a sample which is simple to process.

Description

Gonococcus nucleic acid detection kit and gonococcus nucleic acid detection method

Technical Field

The invention relates to the field of molecular biology detection, and in particular relates to a gonococcus nucleic acid detection kit and a gonococcus nucleic acid detection method.

Background

Gonorrhea is a widely prevalent sexually transmitted disease in the world, and the incidence rate of gonorrhea is the top of China among sexually transmitted diseases, and the gonorrhea tends to rise in recent years. Gonorrhea is a sexually transmitted disease mainly manifested by purulent infection of the urogenital system caused by gonococcus (NG), and male is mainly manifested by urethritis with frequent micturition, urgent micturition, odynuria and purulent secretion of urethra; women mainly have cervicitis, which is typically manifested by mucopurulent secretion in the cervical orifice and urethral irritation accompanied by urethritis.

However, since gonococci (NG) infection in humans is usually asymptomatic gonorrhea in the early stage, the clinical manifestations are similar to those of infection symptoms such as chlamydia trachomatis, and it is difficult to find timely treatment. The untimely treatment can cause serious urogenital tract diseases, and particularly female patients often cause pelvic inflammatory diseases and secondary infertility. Meanwhile, asymptomatic female gonorrhea carriers play an important role in spreading gonococcal (NG) infection and are also the main cause of neonatal gonorrhea. Therefore, the establishment of a method for accurately and quickly diagnosing gonococcal (NG) infection is of great significance.

The traditional gonococcus (NG) detection methods comprise smear detection, culture detection and antigen detection, and different detection methods respectively have advantages and disadvantages. The real-time fluorescence quantitative PCR technology which is a nucleic acid detection technology with higher sensitivity and stronger specificity is developed by combining the traditional PCR technology and the spectroscopic technology. At present, a plurality of gonococcus (NG) nucleic acid detection kits based on a real-time fluorescent quantitative PCR technology are introduced at home and abroad, but most of the existing gonococcus (NG) nucleic acid detection kits have the defect of poor specificity, have strict requirements on samples, and need to be subjected to series of complicated treatments.

Disclosure of Invention

The invention provides a gonococcus nucleic acid detection kit and a gonococcus nucleic acid detection method, and aims to solve the problems of poor kit specificity and complicated sample treatment method in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a gonococcus nucleic acid detection kit is characterized by comprising an upstream primer, a downstream primer and a probe, wherein the sequences of the upstream primer, the downstream primer and the probe are as follows: an upstream primer: AAAGCCTTCCCAACTCG; a downstream primer: CGCCCAAATGGTTACTTC; and (3) probe: 5'-FAM-TGGTCGTCTATCCGTTCCGT-BHQ1-3'; wherein x is a fluorescence reporter group, and y is a fluorescence quencher group.

Preferably, x is a FAM group and y is a BHQ1 group.

Preferably, the gonococcal nucleic acid detection kit further comprises a DNA polymerase, and the DNA polymerase is a hot start DNA polymerase.

Preferably, the gonococcus nucleic acid detection kit further comprises a PCR reaction solution, wherein the PCR reaction solution comprises: reaction buffer, dNTPs, mgCl ₂ 。

Preferably, the gonococcus nucleic acid detection kit further comprises: a first nucleic acid extraction solution: 20% -30% of polyethylene glycol and 0.1M of sodium chloride; second nucleic acid extraction solution: 0.1 to 0.5 percent of Tween 20 and 0.1 to 0.2 percent of sodium dodecyl sulfate.

Preferably, the PCR amplification system is included, and the PCR amplification system comprises 33.4 mu L of PCR reaction liquid and 0.6 mu L of DNA polymerase.

Preferably, the PCR reaction solution comprises 1 × reaction buffer, 0.3mM dNTPs,1mM MgCl ₂ A solution, wherein the upstream primer and the downstream primer are 0.2 mu M each, and the probe is 0.2 mu M; the DNA polymerase is 3.0U.

A gonococcus nucleic acid detection method comprises the following steps: extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample; adding the upstream primer, the downstream primer and the probe to perform PCR amplification reaction by taking the extracted nucleic acid sample as a template; the fluorescent signal is detected during the PCR amplification reaction.

Preferably, in the PCR amplification reaction, the procedure of the PCR amplification reaction is: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15s,40 cycles; 52 ℃ annealing 45s,52 ℃ extension 45s,40 cycles.

Compared with the prior art, the invention has the beneficial effects that:

the gonococcus nucleic acid detection kit and the gonococcus nucleic acid detection method provided by the invention have strong specificity to the gonococcus nucleic acid even if the sample treatment process is simple, the detection method consumes less time, the gonococcus nucleic acid in unknown samples such as genital secretion and the like can be quickly and accurately detected, no cross reaction is caused with other pathogens which can be sexually transmitted and easily cause the same or similar clinical symptoms, and the problems of complicated sample treatment method and poor kit specificity in the prior art are solved. In addition, the kit has high sensitivity, the minimum detection limit is 50 copies/mu L, and the detection range is 50 copies/mu L-5 multiplied by 10 ⁵ copies/mu L, and wide detection range.

Drawings

FIG. 1 is a graph showing the amplification of negative samples detected by the gonococcal nucleic acid detection kit provided in the example of the present invention;

FIG. 2 is a graph showing the amplification of positive samples detected by the gonococcal nucleic acid detection kit provided in the example of the present invention;

FIG. 3 is a graph showing the amplification curve of the accuracy test of the gonococcal nucleic acid detecting kit provided in the example of the present invention;

FIG. 4 is a graph showing the amplification curve of the specific assay (enterprise negative reference) of the gonococcal nucleic acid test kit provided in the example of the present invention;

FIG. 5 is a graph showing the amplification curve of the specific assay (other microorganisms) for the gonococcal nucleic acid detecting kit provided in the examples of the present invention;

FIG. 6 is a graph showing the precision experimental amplification curve of the gonococcus nucleic acid detection kit provided in the examples of the present invention;

FIG. 7 is a graph of the experimental amplification curve for the lowest detection limit of the gonococcal nucleic acid detection kit provided in the examples of the present invention;

FIG. 8 is a standard curve chart of the gonococcus nucleic acid detection kit provided in the examples of the present invention.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example one preparation of primers and probes

Analyzing according to sequences of 8 different standard strains such as gonococcus A, B, C, D, E, G, J, P, selecting a common and highly conserved sequence of each strain, and designing specific primers and fluorescent probes capable of detecting 8 different gonococcus strains according to the screened sequences, wherein the sequences are respectively as follows: an upstream primer: AAAGCCTTCCCAACTCG; a downstream primer: CGCCCAAATGGTTACTTC; and (3) probe: 5'-x-TGGTCGTCTATCCGTTCCGT-y-3'; wherein, x of the probe is a fluorescence reporter group, and y of the probe is a fluorescence quenching group.

Further, x of the probe is a FAM group, and y of the probe is a BHQ1 group. It is understood that, in other embodiments, the fluorescent reporter group labeled on each detection probe is not limited to the above, as long as the fluorescent reporter groups labeled at both ends are capable of fluorescence energy resonance transfer, and the fluorescent reporter groups labeled on the detection probes corresponding to different microorganisms are different.

The above primers and probes were synthesized by the agency of Venetian Biotechnology, shanghai, inc., and the synthesized oligonucleotide dry powder was dissolved in sterile 1 XTE (pH 8.0) to 100. Mu.M and stored at-20 ℃.

EXAMPLE two A kit for gonococcal nucleic acid detection

The embodiment provides a kit for detecting nucleic acid of gonococcus, which comprises the following components:

(1) A first nucleic acid extraction solution: 20% -30% of polyethylene glycol and 0.1M of sodium chloride.

(2) A second nucleic acid extraction solution: 0.1 to 0.5 percent of Tween 20 and 0.1 to 0.2 percent of sodium dodecyl sulfate.

(3) PCR reaction solution: reaction buffer, dNTPs (dATP, dTTP, dGTP, dCTP), mgCl ₂ And (3) solution.

(4) A DNA polymerase.

(5) Positive control: a plasmid containing a target gene sequence.

(6) Negative control: DNase-free ultrapure water.

Preferably, the DNA polymerase is a hot start DNA polymerase because it reduces the hot start time in the early stages of the reaction and reduces the formation of secondary DNA structures. It is understood that in other embodiments, the DNA polymerase is not limited to a hot start DNA polymerase, as long as the DNA polymerase is capable of DNA polymerization.

The PCR reaction system was 40. Mu.L, and contained 33.4. Mu.L of the PCR reaction solution and 0.6. Mu.L of DNA polymerase. Wherein the PCR reaction solution comprises ultrapure water, 1 Xreaction buffer solution, 0.3mM dNTPs and 1mM MgCl ₂ Solutions of0.2. Mu.M forward primer, 0.2. Mu.M reverse primer and 0.2. Mu.M probe; 3.0U of DNA polymerase.

The completed kit, assembled according to the above components, was stored at-20 ℃.

EXAMPLE three gonococcal nucleic acid detection method

The embodiment also provides a gonococcus nucleic acid detection method, which comprises the following steps:

(1) Extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample;

(2) Adding the prepared upstream primer, downstream primer and probe to the extracted nucleic acid sample as template for PCR amplification reaction;

(3) The fluorescent signal is detected during the PCR amplification reaction.

The procedure of the PCR amplification reaction can be specifically determined and adjusted according to the buffer salt ion concentration and the length and nucleotide composition of denatured nucleic acid, reaction characteristics and nucleic acid length, and preferably, the PCR amplification reaction can be performed according to the following procedure: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15s,40 cycles; 52 ℃ annealing 45s,52 ℃ extension 45s,40 cycles.

Example four gonococcus nucleic acid detection kit for detecting unknown samples such as genital secretion

The specific operation steps are as follows:

1. sample sampling and processing

1) Sample sampling

Male: the swab is put into the urethra of the male for 2 to 4cm, kept for a plurality of seconds, rotated for 2 weeks and then taken out.

Female: washing off exocervical secretions by using a sterile normal saline cotton ball, replacing a sampling swab, extending into the cervix for 1-2 cm, staying for several seconds, rotating for 2 weeks, and taking out.

2) Sample processing

The swab after sampling was placed in a sampling tube containing 1.0mL of sterile physiological saline for thorough rinsing, squeezed on the tube wall and discarded. And (3) sucking 200 mu L of sample rinsing liquid into a 1.5mL EP tube, adding 50 mu L of first nucleic acid extracting solution, centrifuging at 12000rpm for 5 minutes, then sucking and removing supernatant, adding 40 mu L of second nucleic acid extracting solution, performing vortex oscillation and uniform mixing, incubating at 100 ℃ for 10 minutes, cooling, centrifuging at 12000rpm for 1 minute, and taking supernatant as a template for subsequent PCR amplification. The negative control of the kit is performed simultaneously with the sample extraction.

2. Reagent preparation

According to the number of tubes n of the PCR reaction tube (n = number of samples +1 tube blank +1 tube negative control +1 tube positive control), 33.4. Mu.L × n of the PCR reaction solution and 0.6. Mu.L × n of the DNA polymerase in the kit were taken. After fully mixing, sequentially packaging 34 μ L into each PCR amplification reaction tube, then respectively adding 6 μ L of positive control, negative control and sample DNA into each PCR amplification reaction tube, closing the tube cap, and transferring to a PCR amplification chamber.

3.PCR amplification

1) Placing the PCR reaction tube into a real-time fluorescent quantitative PCR instrument, and setting the name of a sample;

2) Fluorescence channel selection: selecting a FAM channel (Reporter: FAM, quencher: none);

3) PCR reaction conditions were set according to Table 1, after the setting was completed, a PCR amplification program was run, and fluorescence was collected during annealing and extension.

TABLE 1 PCR amplification reaction conditions

4. The result of the detection

After the reaction is finished, the instrument automatically stores the result, can automatically analyze by using software carried by the instrument, can also manually adjust the starting value, the ending value and the threshold value of the baseline to analyze, then records the Ct value and the result of the sample, and obtains the detection result according to the Ct value and the interpretation method of the sample.

1) Quality control product

a. The detected Ct values for the negative and blank controls should be displayed as underdetermined;

b. the Ct value of the positive control should be less than or equal to 28.

The experiment is considered to be effective if the two conditions are met, otherwise, the experiment is invalid and needs to be redone.

2) The result of the detection

1) As shown in fig. 1, the amplification curve is flat, no Ct value shows (undermined) sample, reported as negative for gonococcal nucleic acid detection;

2) As shown in FIG. 2, the amplification curve is S-shaped, and the sample with Ct value less than or equal to 37 reports positive gonococcus nucleic acid detection;

3) The amplification curve is S-shaped, if the Ct value is not less than 37 and not more than 40, the sample is rechecked (the amount of the nucleic acid sample can be doubled), if the Ct value is not less than 37 and not more than 40, the sample is judged to be positive, otherwise, the sample is negative.

Example five accuracy experiments

By using the gonococcus nucleic acid detection kit and the gonococcus nucleic acid detection method, 8 enterprise positive reference products are detected, the experimental data are shown in table 2, and the PCR amplification chart is shown in fig. 3. Amplification curves of 8 enterprise positive reference products are S-shaped, ct values are less than or equal to 37, detection results are positive, and the detection accuracy is high.

TABLE 2 accuracy test data

Name of reference	The result of the detection	Ct value
			P1	Gonococcal positivity	28.61
P2	Gonococcal positivity	24.74
			P3	Gonococcal yangProperty of (2)	22.79
P4	Gonococcal positivity	29.10
			P5	Gonococcal positivity	25.17
P6	Gonococcal positivity	25.86
			P7	Gonococcal positivity	32.04
P8	Gonococcal positivity	28.74

Example six specific experiments were carried out in a six-stage,

by using the gonococcus nucleic acid detection kit and the gonococcus nucleic acid detection method, 8 enterprise negative reference products and microorganisms which are easy to cause the same or similar clinical symptoms and are normally parasitic or easy to be complicated at a sampling part and other microorganisms which have homology with a target gene nucleic acid sequence are detected, the experimental data is shown in table 3, and the PCR amplification map is shown in fig. 4 and 5. As can be seen from FIGS. 4 and 5, the amplification curves are straight lines and have no Ct value, that is, the detection results of 8 enterprise negative reference samples are negative, and the amplification curves have no cross reaction with other microorganisms which easily cause the same or similar clinical symptoms and are normally parasitic or easily complicated at the sampling part, indicating that the specificity is strong.

TABLE 3 specificity test data

/>

Example seven precision experiments

By using the gonococcus nucleic acid detection kit and the gonococcus nucleic acid detection method, 10 sets of repeatability tests are carried out on 2 enterprise reference products (J1 and J2), the experimental data are shown in a table 4, and the PCR amplification map is shown in a figure 6. The detection results are positive and have good repeatability, and the coefficient of variation (CV,%) of the Ct value of precision is less than or equal to 5 percent, which indicates that the precision is high.

TABLE 4 precision experimental data

/>

Example eight lowest detection Limit experiment

Using the above-mentioned gonococcal nucleic acid detection kit and method, 5X 10 pairs of nucleic acids were detected ⁵ copies/μL、5×10 ⁴ copies/μL、5×10 ³ copies/μL、5×10 ² The amplification is carried out on the gonococcus positive plasmids with five concentration gradients of copies/mu L and 50 copies/mu L, and the experimental result is shown in figure 7; and a standard curve chart for quantitative analysis of gonococci was plotted, as shown in fig. 8. According to the experimental result, the minimum detection limit of the gonococcal nucleic acid detection kit is 50 copies/mu L, and the sensitivity is high.

According to the above embodiment, although the sample treatment process is simple, the specific, rapid and accurate detection of gonococci can be achieved by using the gonococcal nucleic acid detection kit and the gonococcal nucleic acid detection method provided by the present inventionNucleic acids are not detected for microorganisms that are susceptible to the same or similar clinical symptoms, normal parasitism or complications at the site of sampling, and other microorganisms that share homology with the nucleic acid sequence of the gene of interest. The invention abandons the fussy sample processing method, solves the problem of poor specificity of the reagent kit in the prior art on the basis of simple sample processing, and leads the gonococcus nucleic acid to be detected accurately, simply, conveniently and quickly. In addition, the kit has high sensitivity, the minimum detection limit is 50 copies/mu L, and the detection range is 50 copies/mu L-5 multiplied by 10 ⁵ copies/. Mu.L, wide detection range.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Shenzhen City Spanish Biotechnology Limited

<120> gonococcus nucleic acid detection kit and gonococcus nucleic acid detection method

<130> QPA1915032CN

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 17

<212> DNA

<213> Artificial sequence

<400> 1

aaagccttcc caactcg 17

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<400> 2

cgcccaaatg gttacttc 18

Claims

1. The gonococcus nucleic acid detection kit is characterized by comprising an upstream primer, a downstream primer and a probe, wherein the sequences of the upstream primer, the downstream primer and the probe are as follows:

an upstream primer: AAAGCCTTCCCAACTCG;

a downstream primer: CGCCCAAATGGTTACTTC;

and (3) probe: 5'-x-TGGTCGTCTATCCGTTCCGT-y-3';

wherein x is a fluorescence reporter group, and y is a fluorescence quencher group;

the x is FAM group, and the y is BHQ1 group;

the gonococcus nucleic acid detection kit also comprises a first nucleic acid extracting solution and a second nucleic acid extracting solution,

first nucleic acid extraction solution: 20% -30% of polyethylene glycol and 0.1M of sodium chloride;

second nucleic acid extraction solution: 0.1% -0.5% of Tween 20 and 0.1% -0.2% of sodium dodecyl sulfate.

2. The gonococcal nucleic acid detection kit of claim 1, further comprising a DNA polymerase, wherein the DNA polymerase is a hot start DNA polymerase.

3. The gonococcal nucleic acid detection kit of claim 1, further comprising a PCR reaction solution, said PCR reaction solution comprising: reaction buffer, dNTPs, mgCl ₂ 。

4. The gonococcal nucleic acid detection kit according to claim 1, wherein said gonococcal nucleic acid detection kit comprises a PCR amplification system, said PCR amplification system comprising 33.4. Mu.L of PCR reaction solution and 0.6. Mu.L of DNA polymerase.

5. The gonococcal nucleic acid detection kit according to claim 4, wherein said PCR reaction solution comprises: 1 × reaction buffer, 0.3mM dNTPs,1mM MgCl ₂ Solution, the upstream primer and the downstream primer are each 0.2. Mu.M, the probe is 0.2. Mu.M, and the DNA polymerase is 3.0U.