Rapid nucleic acid isothermal amplification detection method for gonococcus

Abstract

The invention discloses a rapid nucleic acid isothermal amplification detection method for gonococci. The method comprises the following steps: step 1, designing a gonococci NGporA primer probe combination; step 2, designing a gonococcus NG 16S primer probe combination; 3, designing a reference gene human gene (genome desoxyribonucleic acid) primer probe combination; 4, designing a reference gene human gene (messenger ribonucleic acid) primer probe combination; 5, establishing a rapid nucleic acid isothermal amplification detection system; 6, detecting the sensitivity of the primer probe combination; and 7, carrying out digital polymerase chain reaction amplification on the human gene (genome desoxyribonucleic acid) gene primer probe combination. The method has the remarkable advantages of high sensitivity, strong specificity, good repeatability, simple and convenient operation flow, short detection period, low cost, high accuracy and the like; and the requirements on rapid and accurate detection of gonococcus can be met, the problems of insufficient specificity and cumbersome sample treatment flow of a kit in the prior art are effectively solved, and wide application prospects in the field of gonococcus detection are shown.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection technology in the field of molecular biology, and in particular to a rapid nucleic acid isothermal amplification detection method for gonococci. Background Art

[0002] Sexually transmitted diseases (STDs) are a group of diseases that are primarily transmitted through sexual contact. As a type of infectious disease that poses a serious threat to human health and is widely prevalent, STDs not only harm individual health, but can also have a profound impact on families and society. The damage to human health caused by STDs is multi-dimensional. If not discovered and treated thoroughly in time, infection with STDs can cause damage to reproductive organs and lead to infertility. Some diseases may even damage important organs such as the heart and brain, and in severe cases can lead to death. Certain STDs, such as genital warts and genital herpes, are extremely difficult to cure once infected.

[0003] Internationally, more than 20 infectious diseases transmitted through sexual intercourse are classified as sexually transmitted diseases. According to the classification of the World Health Organization (WHO), sexually transmitted diseases are divided into four levels, including primary STDs such as AIDS, secondary STDs such as syphilis and gonorrhea, tertiary STDs such as condyloma acuminatum and genital herpes, and quaternary STDs such as giardiasis and campylobacteriosis. Secondary STDs are mainly caused by pathogens such as Neisseria gonorrhoeae (NG), Ureaplasma urealyticum (UU), and Chlamydia trachomatis (CT).

[0004] Sexually transmitted diseases spread quickly and are very harmful. Gonorrhea, as one of the sexually transmitted diseases, is widely prevalent worldwide. According to the US Centers for Disease Control and Prevention, there were about 62 million patients infected with gonorrhea in the world in 1997. The incidence of gonorrhea is high, especially among sexually active people, and the incidence is even higher in developing countries. The pathogen of gonorrhea is Neisseria gonorrhoeae (NG), and humans are its only natural host. Gonorrhea generally parasitizes in columnar epithelial cells on the mucosal surface. Its key pathogenic components include pili, outer membrane proteins, proteases and lipopolysaccharides. Gonorrhea mainly infects the urogenital tract, oropharyngeal and anorectal mucosa directly through sexual contact, or infects newborns through the birth canal during delivery. The clinical manifestations are diverse, including simple gonorrhea, pelvic inflammatory disease, oropharyngeal and anorectal fistulas, gonococcal conjunctivitis and disseminated gonorrhea. If not treated in time, it can lead to urethral stenosis, vas deferens and fallopian tube stenosis or even atresia, secondary to ectopic pregnancy and male and female infertility. Therefore, early diagnosis and treatment of gonorrhea is crucial.

[0005] After gonococcal infection, about 1% of male patients and 75% of female patients have no obvious symptoms. Therefore, the accuracy of diagnosing gonococcal infection based on clinical symptoms alone is very low, especially for women. Currently, the main diagnostic methods include smear examination, culture examination and antigen detection. Smear examination is prone to miss diagnosis during the incubation period of gonorrhea, while culture examination is complicated and time-consuming, and there is also a risk of negative results. Therefore, it is particularly necessary to establish an accurate and reliable detection method.

[0006] Traditional gonococcal (NG) detection methods include smear testing, culture testing and antigen testing, each of which has its specific advantages and limitations. In order to improve the detection speed, nucleic acids in pathogens can be directly detected from clinical specimens (such as urethral swabs, vaginal swabs, urine). This method has the advantages of rapidity, accuracy and high sensitivity, and can be used for early diagnosis of gonococci (NG), providing strong technical support for the rapid analysis and control of the epidemic. In particular, nucleic acid isothermal amplification detection has become the simplest and fastest method for gonococcal (NG) detection.

[0007] The gonococcal detection kits currently available on the market generally face the problem of complex operation and the need for unified testing in a central laboratory, and they are in urgent need of optimization and improvement.

[0008] Therefore, providing a method that can overcome the defects of the existing gonococcal detection technology, such as complex operation, long time consumption and insufficient detection accuracy, is a technical problem that the present invention urgently needs to solve. Summary of the invention

[0009] In view of this, the present invention provides a rapid nucleic acid isothermal amplification detection method for gonococci.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions: A rapid nucleic acid isothermal amplification detection method for gonococci comprises the following steps: Step 1: Design of primer-probe combination for gonococcal NGporA The NGporA gene was selected as the target for gonococcal detection. The NGporA gene sequences of different gonococcal strains were systematically compared and analyzed, and the highly conserved regions were selected as the target sequences for primer design, and then specific primers were designed.

[0011] Step 2: Design of Neisseria gonorrhoeae NG 16S primer-probe combination Compare and analyze the NG 16S+23S RNA gene sequences of different strains of gonococci, select the highly conserved region in the NG 16S+23S RNA gene as the target sequence for primer design, and design specific primers; Step 3: Design the primer-probe combination of the internal reference gene human gene (genomic DNA) Select human genes (genomic DNA) as internal reference genes to design specific primers; Step 4: Design the primer-probe combination of the internal reference gene human gene (mRNA) Select human genes (messenger RNA) as internal reference genes and design specific primers; Step 5: Preparation of rapid nucleic acid isothermal amplification detection system The primer-probe mixture was prepared and diluted in a concentration gradient.

[0012] Step 6: Perform primer probe sensitivity test The sensitivity tests of gonococcal NGporA primer probe, gonococcal NG 16S primer probe, human gene genomic deoxyribonucleic acid gene primer probe and human gene messenger RNA primer probe were carried out respectively.

[0013] Step 7: Perform digital polymerase chain reaction amplification on the human gene genomic DNA gene primer probe.

[0014] Preferably, in step 1, the specific primer-probe combination for gonococcal NGporA consists of a forward primer, a reverse primer and a specific probe, and their nucleotide sequences are: forward primer TATGCCGGTTTCCGTGCGTTACGATT, reverse primer AGCCGGAACTGGTTTCATCTGATTACTTTCCA, and probe GATTTTCCGGTTTCAGCGGCAGCATTCAATT[FAM-dT][THF][BHQ1-dT]TCCGAGTCAAAACAGC / C3spacer.

[0015] Preferably, in step 2, the specific primer-probe combination for gonococcal NG 16S consists of a forward primer, a reverse primer and a specific probe, and their nucleotide sequences are: forward primer TCTGAAGAAGGCCTTCGGGTTGTAAAGGA, reverse primer TTCCGATTAACGCTCGCACCCTACGTATTA, and probe TTGTCAGGGAAGAAAAGGCTGTTGCCAA[FAM-dT][THF][BHQ1-dT]CGGCGGCCGATGACGGTA / C3spacer.

[0016] Preferably, in the step 3, a specific primer-probe combination for the internal reference gene human gene genomic deoxyribonucleic acid is formed by a forward primer, a reverse primer and a specific probe, and the nucleotide sequences thereof are: forward primer AAAGAAGTGGGTTTATGGAGGTCCTCTTGTGT, reverse primer TCGGGTCAACGCTAGGCTGGCAGGGGAA, and probe TGGAAAAGGACATTTCCACCGCAAAATGGCCCC[FAM-dT][THF][BHQ-dT]GGTGGTGGCCCCTTC(C3spacer).

[0017] Preferably, in step 4, a specific primer-probe combination for the internal reference gene human gene messenger ribonucleic acid is formed by a forward primer, a reverse primer and a specific probe, and the nucleotide sequences thereof are: forward primer AGGGCTGCTTTTAACTCTGGTAAAGTGGAT, reverse primer TGATGACAAGCTTCCCGTTCTCAGCCTTGA, and probe ACCTCAACTACATGGTTTACATGTTCCAA[FAM-dT](THF)[dT-BHQ1]GATTCCACCCATGGCA(C3spacer).

[0018] Preferably, in step 5, the method for preparing the primer-probe mixture is: centrifuge the primer and probe dry powder at 12,000 revolutions per minute for 1 minute, add enzyme-free water according to the amount of water added to the tube wall to dilute the primer to 100 micromolar, then dilute the 100 micromolar stock solution to 10 micromolar, and prepare the reverse primer and probe in a ratio of 1:1:0.3.

[0019] Preferably, in step 5, the conditions for performing the concentration gradient dilution are: the template is stored at minus 80°C, the commonly used concentration is 10^6 copies, and 5 μl / vial.

[0020] Preferably, in step 6 and step 7, the isothermal amplification reaction conditions are: 39° C., 30 s; 60 cycles; collecting fluorescence signals at 39° C., 30 s, and the fluorescence channel is FAM.

[0021] Compared with the prior art, the present invention has achieved the following technical effects: (1) The present invention relates to a rapid detection technology for gonococcal nucleic acid, which can still show high specificity for gonococcal nucleic acid even when the sample processing process is simplified.

[0022] (2) The detection method of the present invention has a short operation time and can quickly and accurately detect gonococcal nucleic acid in unknown samples such as genital secretions within a few hours. Compared with conventional gonococcal culture and drug sensitivity testing, the detection time is greatly shortened.

[0023] (3) The present invention has no cross-reaction with other pathogens that can be sexually transmitted and cause similar clinical symptoms, effectively solving the problems of cumbersome sample processing methods and insufficient specificity of the test kit in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a result diagram of the sensitivity detection of the gonococcal NGporA primer probe of the present invention; Figure 2 It is the result diagram of the sensitivity detection of Neisseria gonorrhoeae NG 16S primer probe of the present invention; Figure 3 This is a result diagram of the sensitivity detection of the primer probe of the human gene genome deoxyribonucleic acid gene of the present invention; Figure 4 This is a result diagram of the sensitivity detection of the human gene messenger RNA primer probe of the present invention; Figure 5 This is a diagram showing the results of digital polymerase chain reaction amplification of the human gene genome deoxyribonucleic acid gene primer probe in the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Design of primer-probe combination for gonococcal NGporA (1) First, by searching relevant domestic and foreign literature, 16SrRNA, cppB, orf1, opa, and NGporA genes were identified as common targets for gonococcal nucleic acid amplification detection. In view of the excellent sensitivity, specificity, and sequence conservation of the NGporA gene in clinical applications, the present invention selected the NGporA gene as the target gene for gonococcal detection. The sequence information of gonococcal NGporA is shown in Table 1: NGporA sequence sequence Serial Number ORIGIN TGGAAAAGGTAGGTTTTGAAATATCACGAGCGACCATCAGCAGCATCAGCATCAAGAACCCGGACCCAAAAGGGCGCAACATCCGACTGAAAGGCGCACTGTATGAGCAAGATTTCCGATTTGGCGAAGACCTTCGAGCAGACATCACGCACCGAAGCCGCCAGCATAGAGCAACAAACGAAAGCAGACTTAGAGACGTTACGGAAAAATATCAACGAGGCATTGAAGCAAAGCGAGCAGAAAATAACCGCCGATATAAACGCCCGGCAGTTACGCATGAGCAAGGCAGTATTCAAGCCCTATCTGTGGAGCTTGCTAGGTATATCGGCGGCAGGGTTGATAGTCATAGCAGGGCTGTTCATAGCGATATGGAGCGTCAAGAACGAGCTGGACGACTTGAAACAGCAGAGAGCCGAAGCAGAGCGCACCCTAGACCTGTTGGAAACCAAGACCAAAGGTTTGACACTGGAAAATTGCCCAGTCGAGAACAGCAAAGCAACGCGGGTATGCGTAGCGACCGAGAAGCGAATGCTGGACGCGTTAGCGGAATTAGAGAGCAATCACGCAGCAATCGAGTTGAACTACGCCTACGAACACATTACCCACGATTATCCCGCCGACAACGCCAAAATCTTCGACGACTCTCGCGACATCAAAACCCATTCCACCCACCCCCGCCTTTCCGTCGGCTACGATTTCGGCAACTGGCGCATCGCTCTCGATTACGCCCGTTACAATACCTGGAGACATGGCAAGCATATTCACGCAGAACAAACAAAACAATACAATAATGGAATGGTTACTGCCAAAATAGAAACAGACCACACCGACAAAGGCACATTCAAAGCCGAATCCAACTACGGCATCTCTGCCATCTACGATTTCGATACCGGCACCCGCTTCAAACCCTATGTCGGTGCACGGGTTGGTTTAGGCAAAATCAAACACAGCATTGCTGTAGAAGACCAAGTAACCGTTACCATTAACCATATCGTGGAAGGACCGAAAAATGCCCACGACCCCATCCACCAAAGCCGCAGCATCCGCCGCGTGGGCTTCGGCGCGGTAGCAGGCGTAGGCATCGACATCACGCCCAACCTGACCCTGGACACCGGATACCGCTACCACTACTGGGGACACTTGGAAAACACCCACTTCAAAACCCACGAAGCCTCATTGGGCGTGCGCTACCGCTTCTGACTTGGCAGGCGAATTCGGCACGGCGCTCGCCGGTCGCGTTGCGAATCCGTTTGGCGATGCCAGCAAAGCCATTGATCCTTGGGACAGCAATAATAATGTGGCTTCGCAATTGGGTATTTTCAAACGCCACGACGGTATGCCGGTTTCCGTGCGTTACGATTCCCCCGGATTTTCCGGTTTCAGCGGCAGCATTCAATTTGTTCCGAGTCAAAACAGCAAGTCCGCCTATACGCCTGCTACTTTCACGCTGGAAAGTAATCAGATGAAACCAGTTCCGGCTGTTGTCGGCAAGCCGGGGTCGGATGTGTATTATGCCGGTCTGAATTACAAAAATGGCGGCTTTTTCGGAAATTATGCCCTTAAATATGCGAAACACGCCAATGAGGGGCATGATGCTTTCTTTTTGTTCTTGCTCGGCAGAGCGAGTGATACCGATCCATTGAAAAACCATCAGGTACACCGCCTGACGGGCGGCTATGA SEQ ID No.1 (2) The present invention uses the National Center for Biotechnology Information (NCBI) database to systematically compare and analyze the NGporA gene sequences of different gonococcal strains to determine the sites prone to mutation and their corresponding mutation frequencies; (3) Based on the above analysis results, the present invention selects a highly conserved region in the NGporA gene as the target sequence for primer design.

[0027] (4) The online bioinformatics tool Primer Premier 5.0 was used to design specific primers.

[0028] For the specific primer-probe combination of gonococcal NGporA, the fluorescent group selected the universal FAM probe labeling dye, and the quenching group selected the universal BHQ1 probe labeling quencher. The sequence information of the primers and probes is shown in Table 2: Primer or probe Sequence (5'→3') Serial Number NGporA-F1 TATGCCGGTTTCCGTGCGTTACGATT SEQ ID No.2 NGporA-R2 AGCCGGAACTGGTTTCATCTGATTACTTTCCA SEQ ID No.3 NGporA-P3 GATTTTCCGGTTTCAGCGGCAGCATTCAATT[FAM-dT][THF][BHQ1-dT]TCCGAGTCAAAACAGC / C3spacer SEQ ID No.4 Note: In the table, "F" stands for forward primer; "R" stands for reverse primer; "P" stands for probe.

[0029] The above primers and probes were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0030] Example 2: Design of Neisseria gonorrhoeae NG 16S primer-probe combination (1) After extensive searches of domestic and foreign literature, the present invention found that 16SrRNA, cppB, orf1, opa, and NGporA genes are commonly used in nucleic acid amplification detection of gonococci; (2) Through the NCBI database, this study conducted an in-depth analysis of the 16S-23S rRNA gene spacer sequences of different strains of gonococci, aiming to reveal the sites prone to mutation and their mutation frequencies.

[0031] (3) The present invention selects a highly conserved region in the NG 16S+23S (RNA) gene as the target sequence for primer design. The sequence information of gonococcal NG 16S+23S (RNA) is shown in Table 3: NG 16S+23S(RNA) sequence Serial Number ORIGIN TGGCGAGTGGCGAACGGGTGAGTAACATATCGGAACGTACCGGGTAGCGGGGGATAACTGATCGAAAGATCAGCTAATACCGCATACGTCTTGAGAGGGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCCGAGCGGCCGATATCTGATTAGCTGGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTCTGAAGAAGGCCTTCGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAAGGCTGTTGCCAATATCGGCGGCCGATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGGGCGCAGACGGTTACTTAAGCAGGATGTGAAATCCCCGGGCTCAACCCGGGAACTGCGTTCTGAACTGGGTGACTCGAGTGTGTCAGAGGGAGGTGGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCTCCTGGGATAACACTGACGTTCATGTCCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAATTAGCTGTTGGGCAACTTGATTGCTTGGTAGCGTAGCTAACGCGTGAAATTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGTTTTGACATGTGCGGATATTCCTGTACTTGATTCAAATGCGATGTGGGGACGGAGAAGGTTAGGTTGGCAAGCTGTTGGAATAGCTTGTTTAAGCCGGTAGGTGGAAGACTTAGGCAAATCCGGGTTTTCTTAACACCGAGAAGTGATGACGAGTGTCTACGGACACGAAGCAACCGATACCACGCTTCCAGGAAAAGCCACTAAGCTTCAGTTTGAATCGAACCGTACCGCAAACCGACACAGGTGGGCAGGATGAGAATTCTAAGGCGCTTGAGAGAACTCGGGAGAAGGAACTCGGCAAATTGATACCGTAACTTCGGGAGAAGGTATGCCCTCTAAGGTTAAGGACTTGCTCCGTAAGCCCCGGAGGGTCGCAGAGAATAGGTGGCTGCGACTGTTTATTAAAAACACAGCACTCTGCCAACACGAAAGTGGACGTATAGGGTGTGACGCCTGCCCGGTGCCGGAAGGTTAATTGAAGATGTGCAAGCATCGGATCGAAGCCCCGGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCCGCACGAATGGCGTAACGATGGCCACACTGTCTCCTCCCGAGACTCAGCGAAGTTGAAGTGGTTGTGAAGATGCAATCTACCCGCTGCTAGACGGAAAGACCCCGTGAACCTTTACTGTAGCTTTGCATTGGACTTTGAAGTCACTTGTGTAGGATAGGTGGGAGGCTTGGAAGCAGAGACGCCAGTCTCTGTGGAGTCGTCCTTGAAATACCACCCTGGTGTCTTTGAGGTTCTAACCCAGACCCGTCATCCGGGTCGGGGACCGTGCATGGTAGGCAGTTTGACTGGGGCGGTCTCCTCCCAAAGCGTAACGGAGGAGTTCGAAGGTTACCTAGGTCCGGTCGGAAATCGGACTGATAGTGCAATGGCAAAAGGTAGCTTAACTGCGAGACCGACAAGTCGGGCAGGTGCGAAAGCAGGACATAGTGATCCGGTGGTTCTGTATGGAAGGGCCAT SEQ ID No.5 (4) Use the online software Primer Premier 5.0 to design specific primers.

[0032] For the specific primer-probe combination for gonococcal NG 16S, the fluorescent group selected the universal FAM probe labeling dye, and the quenching group selected the universal BHQ1 probe labeling quencher. The sequence information of the primers and probes is shown in Table 4.

[0033] Table 4 Primer and probe sequence information Primer or probe Sequence (5'→3') Serial Number NG 16S-P1-F1 TCTGAAGAAGGCCTTCGGGTTGTAAAGGA SEQ ID No.6 NG 16S-P1-R3 TTCCGATTAACGCTCGCACCCTACGTATTA SEQ ID No.7 NG 16S-P1 TTGTCAGGGAAGAAAAGGCTGTTGCCAA[FAM-dT][THF][BHQ1-dT]CGGCGGCCGATGACGGTA / C3spacer SEQ ID No.8 Note: In the table, "F" stands for forward primer; "R" stands for reverse primer; "P" stands for probe.

[0034] The above primers and probes were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0035] Example 3: Design of primer-probe combination for internal reference gene human gene (genomic DNA) (1) Select human genes (genomic deoxyribonucleic acid) as internal reference genes. The sequence information of human genes (genomic deoxyribonucleic acid) is shown in Table 5: Human genes (genomic DNA) sequence Serial Number ORIGIN ATGCTAGTGCGCAGCGGGTGCATCCCTGTCCGGATGCTGCGCCTGCGGTAGAGCGGCCGCCATGTTGCAACCGGGAAGGAAATGAATGGGCAGCCGTTAGGAAAGCCTGCCGGTGACTAACCCTGCGCTCCTGCCTCGATGGGTGGAGTCGCGTGTGGCGGGGAAGTCAGGTGGAGCGAGGCTAGCTGGCCCGATTTCTCCTCCGGGTGATGCTTTTCCTAGATTATTCTCTGGTAAATCAAAGAAGTGGGTTTATGGAGGTCCTCTTGTGTCCCCTCCCCGCAGAGGTGTGGTGGCTGTGGCATGGTGCCAAGCCGGGAGAAGCTGAGTCATGGGTAGTTGGAAAAGGACATTTCCACCGCAAAATGGCCCCTCTGGTGGTGGCCCCTTCCTGCAGCGCCGGCTCACCTCACGGCCCCGCCCTTCCCCTGCCAGCCTAGCGTTGACCCGACCCCAAAGGCCAGGCTGTAAATGTCACCGGGAGGATTGGGTGTCTGGGCGCCTCGGGGAACCTGCCCTTCTCCCCATTCCGTCTTCCGGAAACCAGATCTCCCACCGCACCCTGGTCTGAGGTTAAATATAGCTGCTGACCTTTCTGTAGCTGGGGGCCTGGGCTGGGGCTCTCTCCCATCCCTTCTCCCCACACACATGCACTTACCTGTGCTCCCACTCCTGATTTCTGGAAAAGAGCTAGGAAGGACAGGCAACTTGGCAAATCAAAGCCCTGGGACTAGGGGGTTAAAATACAGCTTCCCCTCTTCCCACCCGCCCCAGTCTCTGTCCCTTTTGTAGGAGGGACT SEQ ID No.9 (2) Use the online software Primer Premier 5.0 to design specific primers.

[0036] For the specific primer-probe combination of the internal reference gene human gene genomic deoxyribonucleic acid, the fluorescent group selects the universal FAM probe labeling dye, and the quenching group selects the universal BHQ1 probe labeling quencher. The sequence information of the primers and probes is shown in Table 6: Primer or probe Sequence (5'→3') Sequence number GgD 1-2F AAAGAAGTGGGTTTATGGAGGTCCTCTTGTGT SEQ IDNo.10 GgD 1-1R TCGGGTCAACGCTAGGCTGGCAGGGGAA SEQ IDNo.11 Human gene gD Probe1 TGGAAAAGGACATTTCCACCGCAAAATGGCCCC[FAM-dT][THF][BHQ-dT]GGTGGTGGCCCCTTC(C3spacer) SEQ ID No.12 Note: In the table, "F" stands for forward primer; "R" stands for reverse primer; "P" stands for probe.

[0037] The above primers and probes were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0038] Example 4: Design of primer-probe combination of internal reference gene human gene (mRNA) (1) Select human genes (messenger RNA) as internal reference genes. The sequence information of human genes (messenger RNA) is shown in Table 7: Human genes (mRNA) sequence Serial Number ORIGIN GCTCTCTGCTCCTCCTGTTCGACAGTCAGCCGCATCTTCTTTTGCGTCGCCAGCCGAGCCACATCGCTCAGACACCATGGGGAAGGTGAAGGTCGGAGTCAACGGATTTGGTCGTATTGGGCGCCTGGTCACCAGGGCTGCTTTTAACTCTGGTAAAGTGGATATTGTTGCCATCAATGACCCCTTCATTGACCTCAACTACATGGTTTACATGTTCCAATATGATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATCACCATCTTCCAGGAGCGAGATCCCTCCAAAATCAAGTGGGGCGATGCTGGCGCTGAGTACGTCGTGGAGTCCACTGGCGTCTTCACCACCATGGAGAAGGCTGGGGCTCATTTGCAGGGGGGAGCCAAAAGGGTCATCATCTCTGCCCCCTCTGCTGATGCCCCCATGTTCGTCATGGGTGTGAACCATGAGAAGTATGACAACAGCCTCAAGATCATCAGCAATGCCTCCTGCACCACCAACTGCTTAGCACCCCTGGCCAAGGTCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGACCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGTATGACAACGAATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAGTAAGACCCCTGGACCACCAGCCCCAGCAAGAGCACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTAGACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTACCCTGTGCTCAACCA SEQ ID No.13 (2) Use the online software Primer Premier 5.0 to design specific primers.

[0039] For the specific primer-probe combination of the internal reference gene human gene messenger RNA, the fluorescent group selects the universal FAM probe labeling dye, and the quenching group selects the universal BHQ1 probe labeling quencher. The sequence information of the primers and probes is shown in Table 8: Table 8 Primer and probe sequence information Primer or probe Sequence (5'→3') Serial Number Human gene mR 1-3F AGGGCTGCTTTAACTCTGGTAAAGTGGAT SEQ ID No.14 Human gene mR 1-2R TGATGACAAGCTTCCCGTTCTCAGCCTTGA SEQ ID No.15 Human gene mR Probe1 ACCTCAACTACATGGTTTACATGTTCCAA[FAM-dT](THF)[dT-BHQ1]GATTCCACCCATGGCA(C3spacer) SEQ ID No.16 Note: In the table, "F" stands for forward primer; "R" stands for reverse primer; "P" stands for probe.

[0040] The above primers and probes were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0041] Example 5: Preparation of Rapid Nucleic Acid Constant Temperature Amplification Detection System (1) The method for preparing the primer-probe mixture is as follows: centrifuge the primer and probe dry powder at 12,000 rpm for 1 minute, add enzyme-free water according to the amount of water added to the tube wall to dilute the primer to 100 μM, then dilute the 100 μM stock solution to 10 μM, and prepare the primer-probe mixture with the reverse primer and probe in a ratio of 1:1:0.3 for standby use.

[0042] (2) The template is stored at -80°C. The commonly used concentration is 10^6 copies. The template is diluted in a 5 μL tube according to the desired final concentration.

[0043] The dilution method is as follows: Use 1×10^6 copies / μl, take 5 μl + 45 μl (1ng / μl λDNA) and dilute to 10^5 copies / μl; Dilute gradually to 10^3 copies / μl, take 45 μl + 45 μl (1ng / μl λDNA) and dilute to 500 copies / μl; [Take 5 μl and add to the system, this is 2500 copies of template] 500 copies / μl, take 5 μl + 45 μl (1ng / μl λDNA) and dilute to 50 copies / μl; [Take 5 μl and add it to the system, this is 250 copies of template] The other concentration gradient dilution methods are similar. λDNA was purchased from Thermo, specification: 500 μg, 300 ng / μl, item number: SD0011.

[0044] Example 6: Sensitivity detection of Neisseria gonorrhoeae NGporA primer probe (1) DNA fluorescence isothermal amplification reaction system: prepare isothermal amplification reaction solution according to the composition in Table 9, add it to a 1.5 ml centrifuge tube, oscillate and mix for a few seconds, and centrifuge at 3000 rpm for a few seconds. The prepared isothermal amplification reaction solution can be stored at 4°C for 1 hour or stored at -20°C for 4 hours.

[0045] Reagents factory Part Number Specification 50uL corresponds to volume / microliter DNA Kit-EXO Fluorescent Lyophilized Beads Qiankang Gene QK-BA-002 48 reactions / box 2 2× reaction buffer Qiankang Gene QK-BA-001 1.2 ml / vial 25 Enzyme-free water Shanghai Grandview Hotel ST876-500ml 500ml / bottle 12.9 NGporA Primer Probe Mix Shanghai Jierui / 10 micromolar 4.6 NG DNA Template Shanghai Jierui / Different copy numbers 5 Magnesium acetate Qiankang Gene QK-BB-001 120 μl / tube 2.5 Total volume / / / 50 temperature time Number of cycles 39℃ 30min 60 cycles FAM fluorescence, collected every 30 seconds (3) Experimental results: The results of the NG gene experiment are as follows Figure 1 As shown: Using the ABI7500 model, 500 copies and 50 copies of the template were tested respectively. The rapid nucleic acid isothermal amplification detection method of the present invention showed extremely high sensitivity. On the ABI7500 model, the minimum detection limit of the NG gene reached 50 copies. The experimental results show that this method has high sensitivity for the diagnosis of gonococci (NG).

[0046] Example 7: Sensitivity detection of Neisseria gonorrhoeae NG 16S primer probe (1) RNA fluorescence isothermal amplification reaction system: prepare isothermal amplification reaction solution according to the composition in Table 10, add it to a 1.5 ml centrifuge tube, oscillate and mix for a few seconds, and centrifuge at 3000 rpm for a few seconds. The prepared isothermal amplification reaction solution can be stored at 4°C for 1 hour or stored at -20°C for 4 hours.

[0047] Reagents factory Part Number Specification 50uL corresponds to volume / microliter RNA Kit-EXO Fluorescent Lyophilized Beads Qiankang Gene QK-BA-004 48 reactions / box 2 2× reaction buffer Qiankang Gene QK-BA-001 1.2 ml / vial 25 Enzyme-free water Shanghai Grandview Hotel ST876-500ml 500ml / bottle 12.9 NG 16S Primer Probe Mix Shanghai Jierui / 10 micromolar 4.6 NG 16S+23S template Shanghai Jierui / Different copy numbers 5 Magnesium acetate Qiankang Gene QK-BB-001 120 μl / tube 2.5 Total volume / / / 50 temperature time Number of cycles 39℃ 30min 60 cycles FAM fluorescence, collected every 30 seconds (3) Experimental results: The results of the NG 16S gene experiment are as follows: Figure 2 Shown: The machine used is Roche 480, 2500 copies and 500 copies of templates were tested respectively. The rapid nucleic acid isothermal amplification detection method of the present invention showed excellent sensitivity. The Ct value of the NG 16S gene on the Roche 480 model changed in a gradient as the template concentration decreased, and the minimum detection limit was 500 copies. The experimental results confirmed that the method has a high sensitivity for the diagnosis of gonococci (NG).

[0048] Example 8: Sensitivity detection of primer probes for human gene genome DNA (1) DNA fluorescence isothermal amplification reaction system: prepare isothermal amplification reaction solution according to the composition in Table 11, add to a 1.5 ml centrifuge tube, oscillate to mix for a few seconds, and centrifuge at 3000 rpm for a few seconds. The prepared isothermal amplification reaction solution can be stored at 4°C for 1 hour or stored at -20°C for 4 hours.

[0049] Reagents factory Part Number Specification 50uL corresponds to volume / microliter DNA Kit-EXO Fluorescent Lyophilized Beads Qiankang Gene QK-BA-002 48 reactions / box 2 2× reaction buffer Qiankang Gene QK-BA-001 1.2 ml / vial 25 Enzyme-free water Shanghai Grandview Hotel ST876-500ml 500ml / bottle 12.9 Human genomic DNA primer probe mixture Shanghai Jierui / 10 micromolar 4.6 Human genomic DNA template Shanghai Jierui / Different copy numbers 5 Magnesium acetate Qiankang Gene QK-BB-001 120 μl / tube 2.5 Total volume / / / 50 temperature time Number of cycles 39℃ 30min 60 cycles FAM fluorescence, collected every 30 seconds (3) Experimental results: The results of the human gene genome DNA gene experiment are as follows Figure 3 As shown: Using the ABI7500 model, 1 nanogram and 500 picograms of templates were detected respectively. The rapid nucleic acid isothermal amplification detection method of the present invention exhibits excellent sensitivity on the ABI7500 model, and the minimum detection limit of the human gene genome deoxyribonucleic acid gene is 500 picograms.

[0050] Example 9: Sensitivity detection of human gene messenger RNA primer probe (1) RNA fluorescence isothermal amplification reaction system: prepare isothermal amplification reaction solution according to the composition in Table 12, add to a 1.5 ml centrifuge tube, oscillate and mix for a few seconds, and centrifuge at 3000 rpm for a few seconds. The prepared isothermal amplification reaction solution can be stored at 4°C for 1 hour or stored at -20°C for 4 hours.

[0051] Reagents factory Part Number Specification 50uL corresponds to volume / microliter 20 μl RNA Kit-EXO Fluorescent Lyophilized Beads Qiankang Gene QK-BA-004 48 reactions / box 2 2× reaction buffer Qiankang Gene QK-BA-001 1.2 ml / vial 25 Enzyme-free water Shanghai Grandview Hotel ST876-500ml 500ml / bottle 12.9 Human gene messenger RNA primer probe mixture Shanghai Jierui / 10 micromolar 4.6 Human messenger RNA Shanghai Jierui / Different copy numbers 5 Magnesium acetate Qiankang Gene QK-BB-001 120 μl / tube 2.5 Total volume / / / 50 temperature time Number of cycles 39℃ 30min 60 cycles FAM fluorescence, collected every 30 seconds (3) Experimental results: The results of the human gene messenger RNA gene experiment are as follows Figure 4 As shown: Model ABI7500, 2500 copies and 250 copies of templates were detected respectively. The rapid nucleic acid isothermal amplification detection method of the present invention shows excellent sensitivity, and the minimum detection limit of human gene messenger RNA gene on the ABI7500 model is 250 copies.

[0052] Example 10: Digital polymerase chain reaction amplification of the human gene genome DNA primer probe combination (1) Preparation of DNA fluorescence isothermal amplification reaction system: Prepare isothermal amplification reaction solution according to the composition in Table 13, add to a 1.5 ml centrifuge tube, oscillate to mix for a few seconds, and centrifuge at 3000 rpm for a few seconds. The prepared isothermal amplification reaction solution can be stored at 4°C for 1 hour or stored at -20°C for 4 hours.

[0053] Reagents factory Part Number Specification 50 microliters corresponds to volume / microliter DNA Kit-EXO Fluorescent Lyophilized Beads Qiankang Gene QK-BA-002 48 reactions / box 2 2× reaction buffer Qiankang Gene QK-BA-001 1.2 ml / vial 25 Enzyme-free water Shanghai Grandview Hotel ST876-500ml 500ml / bottle 12.9 Human genomic DNA primer probe mixture Shanghai Jierui / 10 micromolar 4.6 Human genomic DNA template Shanghai Jierui / Different copy numbers 5 Magnesium acetate Qiankang Gene QK-BB-001 120 μl / tube 2.5 Total volume / / / 50 (2) Droplet generation conditions Water phase: 20 μl of the above reaction system; Oil phase: 40 μl of droplet oil, the main component of which is oil phase substances such as mineral oil, and commercially available products can be used without limitation.

[0054] The above-mentioned water phase and oil phase were used to generate droplets using Suzhou Qiankang Gene's fully automatic sample processing system, model DN0108. The droplet generation conditions were: 210 seconds, 26 kPa; and an oil-in-water droplet phase was generated.

[0055] temperature time Number of cycles 39℃ 30min 60 cycles FAM fluorescence, collected every 30 seconds The amplified product was photographed with fluorescence, and the exposure time was 200 milliseconds.

[0056] (4) Data statistics and analysis: Count the number of positive and negative reaction units, and use statistical methods to calculate the absolute copy number of the target molecule in the sample. The data analysis of digital polymerase chain reaction is based on Poisson distribution, which assumes that: (a) the target molecules are randomly distributed in the micro-reaction unit; (b) the probability of each target molecule entering the reaction unit is independent. The Poisson distribution formula is as follows: Poisson distribution formula: in, : The probability of k events occurring; : Average number of events (average number of copies of the target molecule in each microreaction unit); : The number of times an event occurs (the number of target molecules in digital PCR).

[0057] In digital PCR, we usually focus on (negative) and (positive) situation.

[0058] Estimation of average copy number By counting the proportion of negative reaction units, the average copy number can be estimated : or in, is the ratio of positive reaction units.

[0059] (5) The amplified products were analyzed using an 8-channel droplet reading system. The experimental results are as follows: Figure 5 As shown, first determine the background fluorescence intensity based on the blank control without template addition, that is, dark droplets represent negative amplification products; set droplets that exceed the background fluorescence intensity as positive droplets, that is, bright droplets represent positive amplification products; measure the fluorescence intensity of each reaction droplet again, and count the number of positive droplets. By counting the number of positive droplets and determining the volume of nucleic acid sample added, the amount of human gene genome deoxyribonucleic acid in the nucleic acid sample can be calculated. The experimental results of digital PCR show the same pattern as the results of qPCR, indicating that the minimum detection limit of human gene genome deoxyribonucleic acid genes is 500 pg.

[0060] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A rapid nucleic acid isothermal amplification detection method for gonococci, characterized in that: The steps include: Step 1: Design of primer-probe combination for gonococcal NGporA The NGporA gene was selected as the target gene for gonococcal detection, the NGporA gene sequences of different gonococcal strains were systematically compared and analyzed, and the highly conserved regions in the NGporA gene were selected as the target sequences for primer design to design specific primers. Step 2: Design of Neisseria gonorrhoeae NG 16S primer-probe combination Through a detailed comparison and analysis of the NG 16S+23S RNA gene sequences of different strains of gonococci, we selected the highly conserved regions as the target sequences for primer design and carried out the design of specific primers. Step 3: Design the primer-probe combination of the internal reference gene human gene (genomic DNA) Select human genes (genomic DNA) as internal reference genes to design specific primers; Step 4: Design the primer-probe combination of the internal reference gene human gene (mRNA) Select human genes (messenger RNA) as internal reference genes and design specific primers; Step 5: Preparation of rapid nucleic acid isothermal amplification detection system Prepare a primer-probe mixed solution and perform concentration gradient dilution; Step 6: Perform primer probe sensitivity test The sensitivity tests of gonococcal NGporA primer probe, gonococcal NG 16S primer probe, human gene genomic DNA primer probe, and human gene messenger RNA primer probe were performed respectively; Step 7: Perform digital polymerase chain reaction amplification on the human gene genomic DNA gene primer probe.

2. A rapid nucleic acid isothermal amplification detection method for gonococci according to claim 1, characterized in that: In the step 1, a specific primer-probe combination for gonococcal NGporA is prepared, which consists of a forward primer, a reverse primer and a specific probe, and the nucleotide sequences thereof are: forward primer TATGCCGGTTTCCGTGCGTTACGATT, reverse primer AGCCGGAACTGGTTTCATCTGATTACTTTCCA, and probe GATTTTCCGGTTTCAGCGGCAGCATTCAATT[FAM-dT][THF][BHQ1-dT]TCCGAGTCAAAACAGC / C3spacer.

3. A rapid nucleic acid isothermal amplification detection method for gonococci according to claim 1, characterized in that: In step 2, we used a specific primer-probe combination for gonococcal NG 16S, including a forward primer, a reverse primer and a specific probe, whose nucleotide sequences were: forward primer TCTGAAGAAGGCCTTCGGGTTGTAAAGGA, reverse primer TTCCGATTAACGCTCGCACCCTACGTATTA, and probe TTGTCAGGGAAGAAAAGGCTGTTGCCAA[FAM-dT][THF][BHQ1-dT]CGGCGGCCGATGACGGTA / C3spacer.

4. A rapid nucleic acid isothermal amplification detection method for gonococci according to claim 1, characterized in that: Step 3 involves a specific primer-probe combination of the internal reference gene human gene genomic deoxyribonucleic acid, which includes a forward primer, a reverse primer and a specific probe, and its nucleotide sequence is listed in detail as follows: forward primer AAAGAAGTGGGTTTATGGAGGTCCTCTTGTGT, reverse primer TCGGGTCAACGCTAGGCTGGCAGGGGAA, probe TGGAAAAGGACATTTCCACCGCAAAATGGCCCC[FAM-dT][THF][BHQ-dT]GGTGGTGGCCCCTTC(C3spacer).

5. A rapid nucleic acid isothermal amplification detection method for gonococci according to claim 1, characterized in that: In step 4, we used a specific primer-probe combination targeting the internal reference gene human gene messenger RNA, which consisted of a forward primer, a reverse primer and a specific probe, and its nucleotide sequence is listed as follows: forward primer AGGGCTGCTTTTAACTCTGGTAAAGTGGAT, reverse primer TGATGACAAGCTTCCCGTTCTCAGCCTTGA, probe ACCTCAACTACATGGTTTACATGTTCCAA[FAM-dT](THF)[dT-BHQ1]GATTCCACCCATGGCA(C3spacer).

6. A rapid nucleic acid isothermal amplification detection method for gonococci according to claim 1, characterized in that: In step 5, the method for preparing the primer-probe mixture is as follows: first, centrifuge the primer and probe dry powder at a speed of 12,000 revolutions per minute for 1 minute. Then, add enzyme-free water according to the amount of water marked on the tube wall to dilute the primer to 100 micromolar, and then dilute the 100 micromolar stock solution to 10 micromolar. Finally, prepare the reverse primer and probe combination in a ratio of 1:1:0.

3.

7. A rapid nucleic acid isothermal amplification detection method for gonococci according to claim 1, characterized in that: In step 5, the conditions for gradient dilution are: the template is stored at -80°C, the commonly used concentration is 10^6 copies, and the volume is 5 μl.

8. A rapid nucleic acid isothermal amplification detection method for gonococci according to claim 1, characterized in that: In step 6 and step 7, the isothermal amplification reaction conditions are: 39° C., 30 s; 60 cycles; collecting fluorescence signals at 39° C., 30 s, and the fluorescence channel is FAM.

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