CN110878369B - Kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technology and application thereof - Google Patents
Kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technology and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting neisseria gonorrhoeae nucleic acid based on an RNA isothermal amplification-gold probe chromatography technology and application thereof. The kit releases pathogen nucleic acid after a collected sample is cracked by cell lysate, and then the pathogen nucleic acid fragments are amplified through reverse transcription and transcription processes under the action of reverse transcriptase and T7RNA polymerase. The amplified RNA product is identified and captured by a specific probe in the detection liquid to form an RNA amplification product-specific probe-gold probe complex, and the complex is fixed on an NC membrane through lateral flow chromatography to form a visible strip, so that the detection of pathogen nucleic acid is realized. The invention has the advantages of no RNA extraction process, no special instrument, no pollution in the actual detection based on RNA isothermal amplification, high sensitivity, strong specificity and simple operation, and makes the neisseria gonorrhoeae nucleic acid detection widely applicable.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technology and application thereof.
Background
Neisseria gonorrhoeae (Neisseria gonorrhoeae, NG) is a pathogen causing gonorrhea, is a strict parasitic bacterium in humans, is often present in cells of purulent secretions of acute urethritis and vaginitis, is shaped like an oval or bean, and has a cell length of 0.6 to 0.8 μm and a cell width of about 0.5 μm. Often arranged in pairs, with flat or slightly concave adjacent faces, like two beans paired together. No flagellum, no sporulation, and gram staining negative.
Neisseria gonorrhoeae is a pathogenic bacterium of gonorrhea, has strict human parasitism, and has strong adaptability and invasiveness to human bodies. In the early stage of neisseria gonorrhoeae infection, the human body has no clinical symptoms, but serious urogenital tract diseases can be caused if diagnosis and treatment are not carried out in time, especially female patients often cause pelvic inflammation or secondary infertility, and timely and accurate diagnosis of neisseria gonorrhoeae infection has become a key for treating gonorrhea. The gonococcal infection can cause suppurative infection of genitourinary system, and can also cause infection outside genitourinary tract, such as proctitis, pharyngitis, conjunctivitis of neonate, etc. The clinical manifestations are urethritis and cervicitis, and typical symptoms are difficult urination, frequent urination, urgent urination, painful urination, discharge of mucus or purulent secretion, etc.
Gonorrhea caused by neisseria gonorrhoeae infection is a common sexually transmitted disease.
The establishment of an early, rapid and accurate method for detecting NG infection is of great significance. In combination with domestic conditions, in experimental diagnosis of urogenital tract NG infection, a classical cell culture method is specific, but has long time consumption, high cost, high technical equipment requirements, inapplicability to processing a large number of samples and low sensitivity; the PCR method can directly detect the NG nucleic acid, has high sensitivity, strong specificity and higher detection speed, has considerable advantages in shortening the detection window period and improving the pathogen detection rate, and is one of the main methods for detecting the NG pathogen. The existing common neisseria gonorrhoeae nucleic acid detection methods are all based on fluorescent PCR, and the methods require complex RNA extraction processes, special PCR amplification conditions, special laboratories and fluorescent quantitative PCR instruments, and are extremely easy to pollute in the detection process, so that the wide application of the methods is limited, and the methods are unfavorable for the popularization and application in some communities and remote hospitals. Therefore, there is still a need to find a simple, rapid, inexpensive and highly sensitive method for detecting NG nucleic acid. The invention combines RNA isothermal amplification technology and colloidal gold chromatography technology, and can realize the detection of neisseria gonorrhoeae nucleic acid in a short time.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technique and application thereof. The kit releases pathogen nucleic acid after a collected sample is cracked by cell lysate, and then the pathogen nucleic acid fragments are amplified through reverse transcription and transcription processes under the action of reverse transcriptase and T7RNA polymerase. The amplified RNA product is identified and captured by a specific probe in the detection liquid to form an RNA amplification product-specific probe-gold probe complex, and the complex is fixed on an NC membrane through lateral flow chromatography to form a visible strip, so that the detection of pathogen nucleic acid is realized. Therefore, the invention has the advantages of no complex RNA extraction process, no special instrument, no pollution in the actual detection based on the characteristic of easy degradation of RNA molecules, high sensitivity, strong specificity and simple operation, and can make the neisseria gonorrhoeae nucleic acid detection widely applicable.
In order to achieve the above object, the present invention adopts the following technical scheme:
in a first aspect, there is provided a kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography techniques, comprising:
1) Amplification reaction solution: containing 40mM Tris-HCl (pH 8.0), 12mM MgCl 2 70mM KCl,15% DMSO,5mM DTT, 1mM each dNTP, 2mM each NTP, 0.2. Mu.M each amplification primer, two pairs of primers are required for amplification: neisseria gonorrhoeae amplification primers, internal quality control 18S primers;
(1) Neisseria gonorrhoeae (PorB gene, a conserved region sequence) amplification primers:
NG-R primer (5 '-3'): TAATCTGACTCACTAACGGGAGACTCAGACTGCGGACTGATCG;
NG-F primer (5 '-3'): TTGGTCGACGTACAAACTAGC;
(2) Amplification primers for internal reference gene (human 18SrRNA sequence of conserved region):
reference-R primer (5 '-3'):
TAATACGACTCACTATAGGGAGACACAGTTATCCAAGTAGGAG；
reference-F primer (5 '-3'): TCCTGCCAGTAGCATATGCT;
when designing the primers, the amplification efficiency of each single primer is high, and different primers have no interference. The 5' ends of the R primers of both pairs of primers are introduced with a T7RNA polymerase promoter sequence.
2) Amplification enzyme: including reverse transcriptase (e.g., AMV or M-MLV), T7RNA polymerase, and Rn aseH.
3) Cell lysate (purchased from Signosis, USA, cat. CL-0001): cells can be lysed to release nucleic acids.
4) Detection liquid: the kit comprises a colloidal gold particle marked nucleic acid probe (gold probe), specific probes of each index and a C-line chromogenic probe, wherein each index specific probe comprises two types, namely a CES series and an LES series, and a plurality of CES series and LES series can be designed, and the specific probes are specifically as follows:
(1) Neisseria gonorrhoeae-specific probes (5 '-3'):
NG-CES1：TACGTCTTGAGAGGGAAttttGCTCGACTTGCCACCGAATA；
NG-LES1：TCGGGCCAAGTGCAATCCttttGCCTCAAAGACGGACGCCTTC T；
NG-LES2：ATCAATGCCTACGATATTttttGCCTCAAAGACGGACGCCTTC T；
(2) Reference specific probe (5 '-3')
Internal reference CES1: TTGGCTGAAGAATCCAACttttATCTGTATAGTGTCTGT;
internal reference CES2: TTGACATGGAGCCTGCGGttttATCTGTATAGTGTCTGT;
internal reference LES1: CTTAATTTGACTCAACACttttCGCAGTGCTCGAGCTCTGAG C;
internal reference LES2: CCTAGAAGCACGTCGTTCGttttCGCAGTGCTCGAGCTCTGA G;
internal reference LES3: AATACAGGACTCTTTCttttCCGCAGTGCTCGAGCTCTGAGC;
(3) C line chromogenic probe (5 '-3')
TCAGATCACTATGTACttttCGCAGTGCTCGAGCTCTGAGC；
(4) Gold probe
The 5' end of the gold probe is modified by sulfhydrylation, and the sequence is as follows:
5’-CCTACTCTGCAGTGCTCCATCGTACGTCTGTCATTTTTGCTCAGAGC TCGAGCACTGCG-3’
5) Test strip: the test strip is fixed on a PVC bottom plate, and a sample pad, an NC film and water absorbing paper are sequentially arranged from left to right; the NC film is provided with a C line (quality control line) and two T lines (detection lines), and the directions from the sample pad to the absorbent paper are NG-T, internal reference-T and C lines respectively (as shown in figure 3); NG-T coated NG coated probe, internal reference-T coated internal reference coated probe, C line coated probe, specific sequences (5 '-3') are:
NG coated probe: ACACCAGCTATAGATAttttACACCAGCTATAGATA;
internal reference coated probe: CAGACACTATACAGATttttCAGACACTATACAGAT;
c line coating probe: GTACATAGTGATCTGAttttGTACATAGTGATCTGA.
The invention provides a method for detecting neisseria gonorrhoeae nucleic acid by using the kit for detecting neisseria gonorrhoeae nucleic acid based on the RNA isothermal amplification-gold probe chromatography technology, which comprises the following steps:
(1) Isothermal amplification of RNA
The detection indexes of the invention are two: neisseria gonorrhoeae nucleic acid and human internal reference genes. A pair of (F/R primers) amplification primers was designed for each index, wherein the 5' end of the R primer carries a T7RNA polymerase promoter. The invention realizes the amplification of each index nucleic acid in the same amplification tube, and specifically comprises the following steps: during amplification, under the action of an R primer with a T7 promoter and reverse transcriptase, converting RNA to be detected into RNA, namely cDNA heterozygote; RNA in cDNA is digested by RNaseH in the amplified enzyme to obtain single-stranded cDNA; synthesizing a second strand under the action of the F primer and the DNA polymerase function of reverse transcriptase to form double-stranded DNA with a T7 promoter; double-stranded DNA with a T7 promoter is transcribed by T7RNA polymerase to produce an RNA molecule product. The transcribed RNA molecular product can enter a cyclic amplification process, firstly, F primer can combine with the transcribed RNA molecular product, and the transcribed RNA is converted into RNA by reverse transcriptase; RNA in cDNA is digested by RNaseH in the amplified enzyme to obtain single-stranded cDNA; the R primer is then bound to the single-stranded cDNA, the second strand is synthesized by the action of the reverse transcriptase DNA polymerase, and is again enriched to form more double-stranded DNA molecules with the T7 promoter, so that more transcription templates are provided for the T7RNA polymerase, and a large amount of RNA molecule products are transcribed by the action of the T7RNA polymerase (as shown in figure 1).
The invention designs an internal quality control gene detection for monitoring the effectiveness of sample collection and the effectiveness of an amplification system. When the sample is collected to be qualified, the sample must contain human abscission cells, the human abscission cells must be detected in detection, when the sample is detected to be negative, the internal quality control should be positive, otherwise, the whole detection needs to be resampled for retesting.
(2) Gold probe chromatography
a, designing a specific probe, a gold probe, a C line chromogenic probe and a coating probe
Specific probes: each index-specific probe is comprised of two types: CES series and LES series, each probe may be designed in multiple pieces. Wherein the CES probe comprises two parts, one end of the CES probe can be specifically combined with amplified RNA products, the other end of the CES probe can be assembled with a coated probe coated on an NC membrane to play a role in fixing the amplified RNA products, and the two parts are linked by 4-5T. Each LES probe also comprises two parts, one end of each LES probe can be specifically combined with the amplified RNA product, the other end of each LES probe can be combined with the gold probe, the LES probe plays a role in linking the color development of the gold probe, and the two parts are linked by 4-5T.
Gold probe: the 5' end of the gold probe is modified by sulfhydrylation, and the sulfhydryl group can form a covalent bond with the colloidal gold particles and is marked on the colloidal gold particles. The gold probe may be bound to one end of the specific probe LES.
Coating a probe: the coated probe is fixed on the NC film and can be combined with one end of the specific probe CES to play a role in fixation. Each coated probe contains two copies, and 4-5T-junctions are used between each copy.
C line chromogenic probe: comprising two parts linked by 4-5T. One end of the probe can be combined with a gold probe, and the other end of the probe can be combined with a C line coated probe coated on an NC film. During chromatography, no matter whether RNA amplification products exist or not, the C-line chromogenic probe can form a C-line chromogenic probe-gold probe complex, and the complex can be captured and intercepted by a C-line coating probe on an NC film during chromatography to form a macroscopic strip. The probe can control the quality of the test strip and the detection liquid, and the chromatography process is correct.
The specific probes are required to be free from crossing among probes with different indexes in the design process, and CES series with gold probes and coated probes are required to be free from crossing so as to ensure the detection specificity.
The CES and LES series of the specific probes are designed in a plurality of strips in order to improve immobilization efficiency and bind more gold probes, thereby improving detection sensitivity.
b, test paper strip detection
The test strip is provided with a detection line and a quality control line, wherein the detection line comprises an NG-T and an internal reference-T, and an NG coated probe coated at the NG-T can be specifically combined with one end of a neisseria gonorrhoeae CES series probe; the internal reference coated probe coated at the internal reference-T can be specifically combined with one end of an internal reference CES series probe. And the C line coating probe coated on the quality control line (C line) can be specifically combined with the C line chromogenic probe. The specific probe CES, the specific probe LES, the gold probe and the specific amplified product of the nucleic acid to be detected are hybridized and then dripped on a test strip for chromatography, the color development of a detection line indicates the existence of the nucleic acid to be detected, and the color development of a quality control line indicates the detection effectiveness (shown in figure 2).
In combination with the principle, the working process of the method for utilizing the kit is described as follows:
(1) Nucleic acid extraction
Samples of urethral or vaginal swabs from patients with suspected disease are collected and the viral RNA molecules are released by lysis of the cell lysate.
(2) Isothermal amplification of RNA
To 17. Mu.L of the amplification reaction solution containing Neisseria gonorrhoeae and the internal reference primer, 2. Mu.L of the nucleic acid extract was added, and the mixture was heated at 95℃for two minutes, preheated at 42℃for 2 minutes, 1. Mu.L of the amplification enzyme was added, and the mixture was amplified at 42℃for 1 hour at constant temperature. If neisseria gonorrhoeae exists in the sample to be detected, a large amount of amplification and enrichment are carried out on the index RNA molecules during amplification.
(3) Test strip chromatography
a, prehybridization
The RNA isothermal amplification product was mixed with detection solutions (including specific probes, gold probes, and C-line chromogenic probes) and prehybridized at 42℃for 10 min. The amplified RNA molecules are complementarily paired with specific probes (including CES-series probes and LES-series probes). One end of CES series probes is hybridized and complementarily paired with RNA molecules, and the other end of CES series probes is combined with coated probes on NC films; one end of the LES series probes is hybridized and complementarily paired with the RNA molecules, and the other end of the LES series probes can be complementarily paired with the gold probes for combination, and a CES probe-RNA molecule-LES probe-gold probe complex can be formed when amplification products exist.
b, chromatographic detection
The prehybridization product is dripped on a test strip sample pad, the prehybridization liquid is chromatographed along the NC film towards the direction of absorbent paper, when the RNA amplified product to be detected exists, a CES probe-RNA molecule-LES probe-gold probe complex is formed, and the CES probe-RNA molecule-LES probe-gold probe complex is intercepted by a coating probe coated on the NC film during chromatography to form a macroscopic strip, which is positive (as shown in figure 4).
If no RNA product to be detected is amplified, a CES probe-RNA molecule-LES probe-gold probe complex is not formed, colloidal gold particles cannot be aggregated at the T line, and macroscopic bands are not formed, which is negative (see FIG. 4).
The C-line chromogenic probe can form a C-line chromogenic probe-gold probe complex no matter whether the RNA product to be detected is amplified or not, the complex can flow forwards along the NC membrane during chromatography, and when the complex reaches the C-line, the complex is combined with a sequence coated at the C-line, so that the complex stays at the C-line to form a macroscopic colored band, and the experimental result is effective (as shown in figure 4).
In a second aspect, the application of the kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technology in preparation of neisseria gonorrhoeae detection reagents is provided.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention can amplify two indexes of neisseria gonorrhoeae and internal reference gene simultaneously in the same tube by the RNA isothermal amplification method, the amplified nucleic acid product is RNA, the RNA is easy to degrade in natural environment, and compared with the PCR method, the amplified DNA has the effect of preventing pollution more easily. The isothermal amplification of RNA is carried out in an environment of 42 ℃, and even one water bath kettle can realize the amplification reaction, so that the requirement of an experimental instrument is reduced to the greatest extent.
2. Meanwhile, the invention ensures that each single primer has high amplification efficiency and different primers have no interference with each other through multiple rounds of tests when designing the primers, and the overall amplification effect is good.
3. The invention introduces the function of specific probe CES series and specific probe LES series bridged molecule components during design, and the two probes successfully combine the amplified probes and the RNA nucleic acid amplified fragments in series to realize the specific detection of the index RNA nucleic acid fragments. The use of the two sets of probes ensures that any one set of probes and the index nucleic acid amplification fragment cannot be successfully immobilized on the NC membrane due to hybridization failure, so that a positive detection result cannot be generated, and the detection specificity is ensured. The detection results of the kit of the invention on 20 other microorganisms listed in table 2 are all negative, and the fact that the kit of the invention has no cross reaction with other microorganisms is proved. More than two probes can be designed for each set of probes, and the design is beneficial to improving the sensitivity of the test strip. The minimum detection limit of the kit of the invention on the PCR kit reference derived from the gonorrhea of Chinese food and drug assay institute is 1 multiplied by 10 2 cfu/mL. The detection sensitivity and specificity of the neisseria gonorrhoeae detection for 320 clinical samples with the diagnosis results related to genital tract infection are higher than those of a commercial neisseria gonorrhoeae detection fluorescent quantitative PCR kit.
4. The invention adopts the RNA isothermal amplification technology and the test strip chromatography technology, which not only applies the characteristic of low requirement on instruments by RNA isothermal amplification, but also successfully fuses the characteristic of rapid colloidal gold. The nucleic acid is detected by the test strip, and the result can be interpreted only for about 10 minutes. The method is also quite simple in operation, has low technical requirements on experimental staff, does not need special instruments and equipment, and is easy to popularize in the base layer and remote rural medical institutions for detecting the neisseria gonorrhoeae nucleic acid.
Drawings
FIG. 1 is a schematic diagram of isothermal amplification of RNA;
FIG. 2 is a schematic diagram of the strip color development;
FIG. 3 is a schematic diagram of a test strip;
FIG. 4 is a schematic diagram of detecting yin and yang;
a: NG negative and internal reference negative;
b: NG negative and internal reference positive;
c: NG positive and internal reference positive;
Detailed Description
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification taken in conjunction with the drawings. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever.
The experimental procedure, which does not specify specific conditions in the following examples, is generally followed by conventional conditions, such as "molecular cloning: the conditions described in laboratory Manual 3 rd edition (New York: cold Spring Harbor laboratory Press, 2005) were followed.
Example 1 preparation of nucleic acid detection test strip
The main raw materials required in preparing the nucleic acid detection test strip are as follows: nitrocellulose membrane (NC membrane), sample pad, absorbent paper, PVC base plate, etc.
1. Spraying a film:
detection line NG-T: can capture and bind NG specific probe CES sequence, 10 mu M NG coated probe, spray film amount: 2-3 mu L/cm;
detection line internal reference-T: CES sequence of the combined reference specific probe can be captured, 10 mu M of reference coated probe is sprayed with the film: 2-3 mu L/cm;
quality control line (C line): the color development probe sequence of the combined C line can be captured, the coating probe of 10 mu M C lines is sprayed with the film: 2-3 mu L/cm;
after film spraying, the film is automatically crosslinked once in an ultraviolet crosslinking instrument, and the film is dried in a clean constant temperature box at 37 ℃ for 2 hours and stored in a dry environment for standby.
2. Test strip assembly
And respectively cutting water absorbing paper with the length of 2cm, a coated NC film and a sample pad, and sequentially fixing the water absorbing paper, the coated NC film and the sample pad on a PVC bottom plate from top to bottom to obtain the detection test paper strip. The assembly structure of the test strip is shown in FIG. 3.
Example 2 sensitivity test
The method comprises the steps of determining the lowest detection limit of a gradient dilution of neisseria gonorrhoeae (from a gonorrhoeae PCR kit reference product of Chinese food and drug verification institute), repeating 3-5 parts of each gradient virus dilution, repeating detection for 20 times, taking the level with a positive detection rate of 90% -95% as the lowest detection limit, and detecting the virus dilution as follows:
NG minimum detection limit detection
TABLE 1.1 test data for the detection of different titres NG
TABLE 1.2NG minimum detection limit experimental results
The detection sensitivity of the kit of the invention is finally determined as follows:
| detection index | Minimum detection limit |
| NG | 1×10 2 cfu/mL |
[ example 3 ] specificity verification
1, test strains
After extracting nucleic acid from different microorganisms, detecting, and verifying the design specificity of the primer and the probe of the kit. The relevant pathogens and titers were as follows:
TABLE 2 specificity verification test Strain information
2 test results
The test results were as follows:
TABLE 3 specificity verification test results
3 conclusion
From the data, the detection results of the kit provided by the invention on the microorganisms are negative, and the fact that the kit provided by the invention has no cross reaction with other microorganisms is proved, so that the kit is high in pathogen detection specificity.
Example 4 verification of clinical samples
1 clinical sample information
320 samples of urethral or vaginal swabs from the first hospital in the Wuhan city were tested, with 189 and 131 samples for male and 131 samples for female, respectively, at 59.06% and 40.94% respectively. Of 452 specimens, patients were aged 67 years at maximum, 16 years, 31.25 years on average, 33.5 years on standard deviation, 29 years on median, and the diagnosis of the patients in the group was correlated with genital tract infection.
2, detection result
When in detection, the kit and a commercial neisseria gonorrhoeae fluorescent quantitative PCR kit are used for simultaneously detecting samples, and the detection results are assembled into a four-grid table as follows:
for inconsistent 5 samples, the 'gene sequencing method' is adopted to re-test, 3 samples are positive, the kit provided by the invention is positive, a commercial neisseria gonorrhoeae fluorescent quantitative PCR kit is used for detecting negative samples, 3 samples of the commercial neisseria gonorrhoeae fluorescent quantitative PCR kit are detected by the re-test result, 2 false positive samples are obvious, and the clinical sample neisseria gonorrhoeae detection sensitivity and specificity of the kit provided by the invention are higher.
Sequence listing
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<120> kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technique and application thereof
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Claims (3)
1. A kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography, the kit comprising:
1) Amplification reaction solution: containing 40mM Tris-HCl, pH 8.0, 12mM MgCl 2 70mM KCl,15%DMSO,5mM DTT each dNTP was 1mM, each NTP was 2mM, each amplification primer was 0.2. Mu.M, and two pairs of primers were required for amplification: neisseria gonorrhoeae amplification primers, internal quality control 18S primers;
(1) Neisseria gonorrhoeae amplification primers:
NG-R primer (5 '-3'): TAATCTGACTCACTAACGGGAGACTCAGACTGCGGACTGATCG;
NG-F primer (5 '-3'): TTGGTCGACGTACAAACTAGC;
(2) Amplification primers of reference gene:
reference-R primer (5 '-3'):
TAATACGACTCACTATAGGGAGACACAGTTATCCAAGTAGGAG；
reference-F primer (5 '-3'): TCCTGCCAGTAGCATATGCT;
2) Amplification enzyme: comprises three, reverse transcriptase, T7RNA polymerase and RnaseH;
3) Nucleic acid extraction reagent: cell lysate;
4) Detection liquid: the kit comprises gold probes, a nucleic acid probe marked by colloidal gold particles, specific probes of each index and a C-line chromogenic probe, wherein each index specific probe is two, namely a CES series and an LES series, and a plurality of CES series and LES series can be designed, and the specific probes are specifically as follows:
(1) Neisseria gonorrhoeae-specific probes (5 '-3'):
NG-CES1：TACGTCTTGAGAGGGAAttttGCTCGACTTGCCACCGAATA；
NG-LES1：TCGGGCCAAGTGCAATCCttttGCCTCAAAGACGGACGCCTTC T；
NG-LES2：ATCAATGCCTACGATATTttttGCCTCAAAGACGGACGCCTTC T；
(2) Reference specific probe (5 '-3')
Internal reference CES1: TTGGCTGAAGAATCCAACttttATCTGTATAGTGTCTGT;
internal reference CES2: TTGACATGGAGCCTGCGGttttATCTGTATAGTGTCTGT;
internal reference LES1: CTTAATTTGACTCAACACttttCGCAGTGCTCGAGCTCTGAG C;
internal reference LES2: CCTAGAAGCACGTCGTTCGttttCGCAGTGCTCGAGCTCTGA G;
internal reference LES3: AATACAGGACTCTTTCttttCCGCAGTGCTCGAGCTCTGAGC; (3) C line chromogenic probe (5 '-3')
TCAGATCACTATGTACttttCGCAGTGCTCGAGCTCTGAGC；
(4) Gold probe
The 5' end of the gold probe is modified by sulfhydrylation, and the sequence is as follows:
5’-CCTACTCTGCAGTGCTCCATCGTACGTCTGTCATTTTTGCTCAGAGC TCGAGCACTGCG-3’
5) Test strip: the test strip is fixed on a PVC bottom plate, and a sample pad, an NC film and water absorbing paper are sequentially arranged from left to right; the NC film is provided with a quality control line C line and two detection lines T lines, and the directions from the sample pad to the absorbent paper are NG-T, internal reference-T and C lines respectively; NG-T coated NG coated probe, internal reference-T coated internal reference coated probe, C line coated probe, specific sequences (5 '-3') are:
NG coated probe: ACACCAGCTATAGATAttttACACCAGCTATAGATA;
internal reference coated probe: CAGACACTATACAGATttttCAGACACTATACAGAT;
c line coating probe: GTACATAGTGATCTGAttttGTACATAGTGATCTGA.
2. The kit of claim 1, wherein the reverse transcriptase is AMV or M-MLV.
3. Use of a kit according to claim 1 or 2 in the preparation of a neisseria gonorrhoeae detection reagent.
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