CN114250319B - Kit for detecting sexually transmitted infection multiple nucleic acids - Google Patents
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Abstract
A kit for detecting sexually transmitted infection multiple nucleic acids relates to a nucleic acid detection technology. The kit for detecting the sexually transmitted infection multiple nucleic acid comprises a sexually transmitted pathogen detection primer and a probe, wherein the sequences of the primer and the probe are shown as SEQ ID No. 1-56. The primer probe group comprises 32 tailing primers, 16 medium probes and 7 universal probes. Based on the melting array technology, the single tube can be used for simultaneously detecting 15 sexually transmitted infectious pathogens, wherein the 15 sexually transmitted infectious pathogens comprise bacteria, viruses, chlamydia, mycoplasma and parasites. Has the advantages of simple operation, low cost, high sensitivity, high specificity and rapid detection. The method has the advantages of avoiding PCR post-treatment while realizing parallel detection of a plurality of targets, along with simple and quick operation, low cost, difficult misjudgment and missed judgment caused by melting point deviation, ensuring high sensitivity and high specificity of the system, no need of special equipment and high clinical applicability.
Description
Technical Field
The invention relates to a nucleic acid detection technology, in particular to a kit for detecting sexually transmitted infection multiple nucleic acids.
Background
Sexually transmitted infections (Sexually transmitted infections, STIs) are understood as meaning infectious infections caused by the sexual route. It is estimated by the world health organization that there are about 100 tens of thousands of people per day who are sexually transmitted diseases. Sexually transmitted diseases spread rapidly and pose a serious threat to the health of the population. In women, although most sexually transmitted infections, such as human papilloma virus (Human papillomavirus, HPV), gonorrhea, chlamydia and other pathogen infections are often asymptomatic, complications such as pelvic inflammatory disease, ectopic pregnancy, infertility, and chronic pelvic pain can be caused.
STIs have jumped to the second most common infectious disease in our country and are not optimistic. Syphilis, gonorrhea, genital tract trachoma chlamydia infection, condyloma acuminatum and genital herpes have been the major epidemic species of STIs. Among STIs in China, syphilis is most commonly seen, and is secondarily caused by gonorrhea, genital tract trachoma chlamydia infection, condyloma acuminatum and genital herpes. By 2013, the incidence rate of syphilis report in China reaches 32.86/10 ten thousand people. There are differences in statistics of the number of people who develop the disease, because the situation varies from country to country. Obvious regional differences exist in the incidence rate of STIs in China. Areas with higher incidence of STIs in the whole country are northwest areas, zhujiang delta, changjiang delta, minjiang areas, northeast three provinces, jinjin areas and Chongqing areas, and areas with lower incidence are North China and the middle-primary areas, northwest areas, southwest areas and the like.
The latency of STIs is short and the infectivity is strong, and the rapid diagnosis is important for finding pathogenic bacteria, guiding clinical medication and controlling the development and the spread of diseases. Currently, there are three main methods for sexually transmitted infection screening: morphology observation, immunodetection, molecular biology. Compared with the traditional detection method, the molecular biological detection method directly detects the characteristic target genes of pathogens, and has the advantages of simple detection method, strong specificity, high sensitivity, rapidness, high flux and the like. In recent years, molecular biological detection has become the mainstay of clinical diagnostic techniques for sexually transmitted infectious pathogens. With the continuous popularization and application of the technology, the method is widely adopted by large and medium hospitals and outpatient clinics of venereal diseases.
Real-time PCR is a PCR technique that uses multiple pairs of primers and probes to amplify multiple target sequences simultaneously in a single reaction tube based on conventional PCR. Since the report of real-time PCR, the method has obvious advantages in the aspects of sensitivity, specificity, accuracy and the like, and has been widely applied to various fields of nucleic acid diagnosis, such as mutation analysis, transgene identification, genetic disease diagnosis and pathogen detection. However, real-time PCR is limited by the channel and can only detect up to 6 pathogens. Whereas specimens collected from STIs patients typically contain multiple pathogens. If multiple tubes are used for multiple detection of infectious pathogens, the amount of specimens required is increased, and the detection cost is increased. Currently, patients infected by multiple mixed sexually transmitted diseases are increasing year by year, and only one pathogen can be detected at a time by using a relatively wide fluorescent quantitative PCR technology, so that multiple PCR reactions are required to be performed on one sample to detect whether one or more pathogens exist, the detection cost is high, and the process is complicated. Studies have shown that clinically, as long as patients with STIs, whether or not they are clinically symptomatic, are often infected with a variety of pathogens, and that detection of one or a few sexually transmitted infectious pathogens has failed to meet the clinical diagnostic requirements. Therefore, a simple, rapid and high-throughput detection technology is urgently needed clinically.
Currently, related nucleic acid detection products (sexually transmitted disease detection kit, manufacturer is Hua Xi hospitals in Sichuan university, fluorescent PCR Chlamydia Trachomatis (CT) detection kit, manufacturer is Shanghai flash crystal molecular biotechnology Co., ureaplasma Urealyticum (UU) nucleic acid detection kit (PCR fluorescence method), manufacturer is Aikang biotechnology (Hangzhou) Co., ureaplasma urealyticum nucleic acid detection kit (RNA isothermal amplification), manufacturer is Shanghai kernel biotechnology Co., CMV detection kit (fluorescent quantitative PCR method), manufacturer is Hangzhou Bo technology Co., three genitourinary tract infectious pathogens combined detection gene chip kit, manufacturer is Kunming cloud Biochemical Co., etc.), but there are no products for simultaneous detection of 15 sexually transmitted infectious pathogens. According to online inquiry and market research, 15 pathogens can not be detected simultaneously by related products in the market, and the pathogens can not be detected comprehensively and accurately clinically.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a kit for detecting multiple sexually transmitted infection nucleic acid, which can realize simultaneous detection of 15 sexually transmitted infection pathogens and overcome the limitations of the prior molecular biology technology, based on a melting array (MELTARRAY) technology.
The kit for detecting the sexually transmitted infection multiple nucleic acid comprises a sexually transmitted pathogen detection primer and a probe, wherein the sequences of the primer and the probe are shown as SEQ ID No. 1-56.
Specifically, the primer probe group of the sexually transmitted infection multiplex nucleic acid detection kit comprises the following 32 tail-added primers, 16 medium probes and 7 universal probes:
the sequence of the universal primer Tag is shown as SEQ ID No. 1;
the sequence of the fluorescent probe P-U1-MB-FAM is shown as SEQ ID NO. 2;
The tailing primer for specifically amplifying the human cytomegalovirus HCMV gene sequence has an upstream primer HCMV-F sequence shown as SEQ ID NO.3 and a downstream primer HCMV-R sequence shown as SEQ ID NO. 4;
The vector probe HCMV-MP for detecting the human cytomegalovirus HCMV gene has a sequence shown in SEQ ID NO. 5;
Tail primer for specifically amplifying HSV-1 gene sequence of herpes simplex virus type 1, wherein the upstream primer HSV1-F sequence is shown as SEQ ID NO.6, and the downstream primer HSV1-R sequence is shown as SEQ ID NO. 7;
The vector probe HSV1-MP for detecting the HSV-1 gene of the herpes simplex virus 1 has a sequence shown as SEQ ID NO. 8;
Tail primer for specifically amplifying HSV-2 gene sequence of herpes simplex virus 2, wherein the upstream primer HSV2-F sequence is shown as SEQ ID NO.9, and the downstream primer HSV2-R sequence is shown as SEQ ID NO. 10;
The vector probe HSV2-MP for detecting the HSV-2 gene of the herpes simplex virus 2 has a sequence shown as SEQ ID NO. 11;
the sequence of the fluorescent probe P-U3-MB-FAM is shown as SEQ ID NO. 12.
The tail primer for specifically amplifying the trichomonas vaginalis TV gene sequence has an upstream primer TV-F sequence shown as SEQ ID NO.13 and a downstream primer TV-R sequence shown as SEQ ID NO. 14;
The vector probe TV-MP for detecting trichomonas vaginalis TV gene has a sequence shown as SEQ ID NO. 15;
the primer for adding tail of specific amplification ureaplasma urealyticum UU gene sequence, wherein the upstream primer UU-F sequence is shown as SEQ ID NO.16, and the downstream primer UU-R sequence is shown as SEQ ID NO. 17;
the vector probe UU-MP for detecting the ureaplasma urealyticum UU gene has a sequence shown as SEQ ID NO. 18;
The sequence of the fluorescent probe P-U2-MB-ROX is shown as SEQ ID NO. 19.
The primer for specifically amplifying the tailing of the HD gene sequence of the haemophilus ducreyi has an upstream primer HD-F sequence shown in SEQ ID NO.20 and a downstream primer HD-R sequence shown in SEQ ID NO. 21;
the vector probe HD-MP for detecting the Hd gene of the haemophilus ducreyi has a sequence shown in SEQ ID No. 22;
the tail primer for specifically amplifying the neisseria gonorrhoeae NG gene sequence has an upstream primer NG-F sequence shown as SEQ ID NO.23 and a downstream primer NG-R sequence shown as SEQ ID NO. 24;
The vector probe NG-MP for detecting the neisseria gonorrhoeae NG gene has a sequence shown as SEQ ID NO. 25;
The sequence of the fluorescent probe P-U1-MB-LNA-ROX is shown as SEQ ID NO. 26.
The primer for specifically amplifying the tail of the treponema pallidum TP gene sequence has an upstream primer TP-F sequence shown as SEQ ID NO.27 and a downstream primer TP-R sequence shown as SEQ ID NO. 28;
The intermediate probe TP-MP for detecting treponema pallidum TP gene has a sequence shown in SEQ ID NO. 29;
The tailing primer for specifically amplifying the Chlamydia trachomatis CT gene sequence has an upstream primer CT-F sequence shown as SEQ ID NO.30 and a downstream primer CT-R sequence shown as SEQ ID NO. 31;
The vector probe CT-MP for detecting the Chlamydia trachomatis CT gene has a sequence shown in SEQ ID NO. 32;
tail primer for specific amplification of genital mycoplasma MG gene sequence, its upstream primer MG-F sequence is shown as SEQ ID NO.33, and its downstream primer MG-R sequence is shown as SEQ ID NO. 34;
the vector probe MG-MP for detecting the mycoplasma genitalium MG gene has a sequence shown as SEQ ID NO. 35;
The sequence of the fluorescent probe P-U2-MB-CY5 is shown by SEQ ID NO. 36.
The tail primer for specifically amplifying the gene sequence of the micro ureaplasma UP has an upstream primer UP-F sequence shown as SEQ ID NO.37 and a downstream primer UP-R sequence shown as SEQ ID NO. 38;
The medium probe UP-MP for detecting the gene of the ureaplasma minutissimum has a sequence shown as SEQ ID NO. 39;
Tail primer for specific amplification of granuloma capsular bacillus GI gene sequence, its upstream primer GI-F sequence is shown in SEQ ID NO.40, and its downstream primer GI-R sequence is shown in SEQ ID NO. 41;
The vector probe GI-MP for detecting the GI gene of the granulomatous capsular bacillus has a sequence shown as SEQ ID NO. 42;
The tail primer for specifically amplifying the herpes zoster virus VZV gene sequence has an upstream primer VZV-F sequence shown as SEQ ID NO.43 and a downstream primer VZV-R sequence shown as SEQ ID NO. 44;
the vector probe VZV-MP for detecting the herpes zoster virus VZV gene has a sequence shown as SEQ ID NO. 45;
The sequence of the fluorescent probe P-U3-MB-CY5 is shown by SEQ ID NO. 46.
The primer for specifically amplifying the tailing of the human mycoplasma MH gene sequence has an upstream primer MH-F sequence shown as SEQ ID NO.47 and a downstream primer MH-R sequence shown as SEQ ID NO. 48;
the base sequence of the vector probe MH-MP used for detecting the human mycoplasma MH gene is shown as SEQ ID NO. 49;
The tailing primer for specifically amplifying the human herpesvirus 8-type HHV-8 gene sequence has the upstream primer HHV8-F sequence shown as SEQ ID NO.50 and the downstream primer HHV8-R sequence shown as SEQ ID NO. 51;
the vector probe HHV8-MP for detecting the human herpesvirus 8-type HHV-8 gene has the sequence shown in SEQ ID NO. 52;
the sequence of the fluorescent probe P-U1-MB-HEX is shown as SEQ ID NO. 53.
The tailing primer for specifically amplifying the humanized RPP30 gene sequence has an upstream primer RPP30-F sequence shown as SEQ ID NO.54 and a downstream primer RPP30-R sequence shown as SEQ ID NO. 55;
The vector probe RPP30-MP for detecting the human RPP30 gene has a sequence shown in SEQ ID NO. 56.
The concentration ratio of the upstream primer to the downstream primer to the vector probe in each primer group is 1:1:10.
The kit for detecting the sexually transmitted infection multiple nucleic acid adopts a single tube four-channel for synchronously detecting 15 sexually transmitted infectious pathogens, wherein the 15 sexually transmitted infectious pathogens comprise bacteria, viruses, chlamydia, mycoplasma, parasites and the like.
The bacteria include Treponema Pallidum (TP), neisseria Gonorrhoeae (NG), haemophilus Ducreyi (HD), and bacillus granulomatous capsular bacterium (GI); the virus comprises herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human Cytomegalovirus (HCMV), herpes zoster virus (VZV), and human herpes virus type 8 (HHV-8); the chlamydia and mycoplasma comprise Chlamydia Trachomatis (CT), ureaplasma Urealyticum (UU), tiny Ureaplasma (UP), mycoplasma Genitalium (MG) and Mycoplasma Hominis (MH); the parasites include Trichomonas Vaginalis (TV) and the like.
The kit for detecting the sexually transmitted infection multiple nucleic acid also comprises a PCR reaction solution, wherein the PCR reaction solution comprises a PCR buffer solution, dNTPs (containing dUTP), mgCl 2, SSB, taq01 enzyme and UNG enzyme.
Further, the kit also comprises a positive quality control product and a negative quality control product, wherein the positive quality control product is a cloning plasmid, and the negative quality control product is sterilized purified water.
Sexually transmitted infectious pathogens and conditions
The sexually transmitted pathogen detection kit can realize single-tube simultaneous detection of 15 sexually transmitted infectious pathogens based on a melting array technology, and has the advantages of simplicity and convenience in operation, low cost, high sensitivity, high specificity and rapid detection. The invention can realize parallel detection of a plurality of targets, simultaneously avoid post-PCR treatment, has simple and rapid operation, low cost, is not easy to cause misjudgment and missed judgment due to melting point deviation, ensures high sensitivity and high specificity of the system, does not need special equipment, and has high clinical applicability.
Compared with the prior art, the invention has the following beneficial effects:
(1) The primers of the invention are optimized by a large number of tests, and detection primers for efficient and stable amplification are screened. Through specific sequence design of the primer, a section of universal primer sequence is added at the 5' end of the specific primer, so that the detection sensitivity and the specificity of the primer are improved;
(2) The medium probe provided by the invention is used for screening the detection probe with strong specificity through optimization of a large number of tests. The specific sequence design of the probe is combined with the adjustment of the length and the position of the probe, so that the detection specificity and the accuracy of the medium probe are improved;
(3) The invention provides a kit for detecting multiple nucleic acids of 15 sexually transmitted infectious pathogens, and establishes a method for detecting nucleic acids based on a melting array technology, and a plurality of target sequences can be detected simultaneously in a single reaction tube. The invention can accurately detect 15 pathogens in one test, has large detection flux, can accurately determine which pathogen the patient infects at one time, provides reference basis for judging the illness state, improves the detection efficiency and further reduces the cost.
Drawings
FIG. 1 shows a characteristic melting peak curve of a kit according to an embodiment of the present invention;
FIG. 2 is a typical graph of the detection results of clinical specimens (50 specimens for a single experiment) according to an embodiment of the present invention;
FIG. 3 is a graph showing the detection results of a typical positive clinical specimen according to an embodiment of the present invention. Here, fig. a, B, C, and D show the detection results of the specimen a, B, C, and D, respectively. The solid black line indicates the pathogen detected by the positive specimen, and the dashed black line indicates the negative control without template.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. On the contrary, the invention is intended to cover any alternatives, modifications, equivalents, and variations as may be included within the spirit and scope of the invention as defined by the appended claims. Further, in the following detailed description of the present invention, certain specific details are set forth in order to provide a thorough understanding of the present invention. The present invention will be fully understood by those skilled in the art without the details described herein.
1. Primer and probe design
The invention selects the conserved sequence of each pathogen as an amplification region, designs a specific primer and a probe, and realizes single-tube four-channel qualitative detection of 15 sexually transmitted infectious pathogens by combining a fluorescent probe melting curve technology based on multiplex PCR. The system comprises 32 tailing primers, 16 mediator probes and 7 universal probes (wherein the primer pair of the internal reference gene and the mediator probes are also contained).
The 32 tailing primers comprise an upstream primer HCMV-F and a downstream primer HCMV-R for an HCMV target gene, an upstream primer HSV1-F and a downstream primer HSV1-R for an HSV-1 target gene, an upstream primer HSV2-F and a downstream primer HSV2-R for an HSV-2 target gene, an upstream primer TV-F and a downstream primer TV-R for a TV target gene, an upstream primer UU-F and a downstream primer UU-R for a UU target gene, an upstream primer HD-F and a downstream primer HD-R for an HD target gene, an upstream primer NG-F and a downstream primer NG-R for an NG target gene, an upstream primer TP-R for a TP target gene, an upstream primer CT-F and a downstream primer MG-R for a CT target gene, an upstream primer MG-F and a downstream primer MG-R for a TV target gene, an upstream primer UU-F and a downstream primer UU-R for a UU target gene, an upstream primer UP-F and a downstream primer UP-R for a UP gene, an upstream primer UP-F and a downstream primer, an upstream primer HH-R for a ZMH-R for a target gene, an upstream primer HH-R for a ZMH-8 and a target gene, an upstream primer;
The 7 universal probes comprise a fluorescent probe P-U1-MB-FAM, a fluorescent probe P-U3-MB-FAM, a fluorescent probe P-U2-MB-ROX, a fluorescent probe P-U1-MB-LNA-ROX, a fluorescent probe P-U2-MB-CY5, a fluorescent probe P-U3-MB-CY5 and a fluorescent probe P-U1-MB-HEX;
The 16 medium probes comprise medium probes HCMV-MP, medium probes HSV1-MP, medium probes HSV2-MP, medium probes TV-MP, medium probes UU-MP, medium probes HD-MP, medium probes NG-MP, medium probes TP-MP, medium probes CT-MP, medium probes MG-MP, medium probes UP-MP, medium probes GI-MP, medium probes VZV-MP, medium probes MH-MP, medium probes HHV8-MP and medium probes RPP30-MP;
In the construction process, 3 pairs of primer pairs are respectively designed for each detection object in the single-system construction process in order to ensure the specificity and the amplification efficiency of multiplex PCR amplification, and the primer pairs with high amplification efficiency and nearly consistent amplification efficiencies of different detection objects are selected as single optimal primer pairs by optimizing the combination of every two of the detection objects; and in the process of establishing a multiple system, a primer pair which does not generate nonspecific amplification and has the best amplification efficiency is selected. Since the multiplex system contains a plurality of primers, a plurality of primer dimers are easily formed, and thus non-specific amplification is generated, and thus amplification efficiency is lowered. In order to reduce primer dimer and make the detection sensitivity of the system reach single copy level, the invention introduces a homologous tag auxiliary-primer-free dimer (HAND) system to carry out multiple equivalent specific amplification, thereby realizing target enrichment.
The sequence of the universal primer Tag of the embodiment of the invention is SEQ ID No.1. The sequences of the designed primers, the vector probes and the fluorescent probes are shown in the attached table 1.
Table 1 fifteen PCR melting curve analysis system detected target genes, primers and probes thereof
Note that: the "+" base is the modified base of the locked nucleic acid (Locked Nucleic Acid, LNA).
2. Sample requirement
1) Urine: 1mL of morning urine is taken in a sterile centrifuge tube of 1.5mL, the supernatant is centrifuged off, and the sediment is taken as a specimen. The specimen can be used for testing immediately, and can be stored at-20 ℃ to be tested. The shelf life is 6 months.
2) And (3) swab: sterile physiological water cotton ball washing, removing urinary meatus and external secretion of cervix, wiping urinary tract and genital tract secretion by using disposable sterile cotton swab dipped with physiological saline, and then using the secretion on the cotton swab for physiological water washing and uniformly mixing. The specimen can be used for testing immediately, and can also be stored at-20 ℃ to be tested.
3. Extraction of genomic DNA
1) 1ML of sterilized normal saline is added into a sample test tube, and the sample test tube is fully and uniformly shaken;
2) Transferring all liquid into a 1.5mL centrifuge tube, and centrifuging at 12,000rpm for 5min;
3) Removing supernatant, adding sterilized normal saline 1mL into the precipitate, shaking and mixing well, and centrifuging at 12,000rpm for 5min;
4) Removing supernatant, adding 50uL of nucleic acid extract into the precipitate, mixing well, centrifuging at 12,000rpm for 10min in a water bath at 99 ℃ for 10min, and taking 5uL of supernatant for PCR amplification.
5) For urine specimens, shake the urine, take 1mL to 1.5mL centrifuge tube, centrifuge at 12,000rpm for 5min, then discard the supernatant, mix the precipitate with 1mL sterile saline, centrifuge at 12,000rpm for 5min. The rest steps are the same as those of the step 4).
4. Multiplex real-time PCR amplification reaction system
The multiple real-time PCR reaction system finally determined by optimizing the buffer type, the Mg 2+ dosage, dNTPs and UNG enzyme, the primer proportion, the primer dosage, the probe dosage, the DNA polymerase and the like in the PCR reaction is shown in Table 2.
TABLE 2 component content of multiplex real-time PCR
| PCR reaction solution composition | Dosage (mu L) | 1 Person |
| ddH2O | Complement of | |
| 10×Taq01 buffer | 2.50 | 1× |
| 25mM MgCl2 | 7.00 | 7mM |
| 25mM dNTPs:dUTP | 0.25 | 0.25mM |
| 100μM Tag | 0.20 | 0.80μM |
| 50 Mu M fluorescent probe | 0.02 | 0.04μM |
| 100 Mu M tailing primer | 0.01～0.02 | 0.04～0.08μM |
| 50 Mu M medium probe | 0.02～0.04 | 0.04～0.08μM |
| 10-4SSB | 0.10 | |
| 6U/. Mu.L Taq01: UNG enzyme (20:1) | 0.50 | 2.0U |
| Total amount of | 20 |
Note that: to the reaction tube, 5. Mu.L of the treated sample, 5. Mu.L of the positive quality control, 5. Mu.L of the negative quality control (TE solution or purified water) and 25. Mu.L of the total reaction volume were added, respectively.
5. Condition setting for multiplex real-time PCR amplification
The finally determined multiplex real-time PCR amplification reaction conditions are shown in Table 3 through comparative optimization of a large number of experiments.
TABLE 3 multiplex real-time PCR amplification procedure
Note that: a Fluorescence signals of FAM, ROX, CY, HEX channels were acquired during the annealing extension phase.
b After the completion of the PCR amplification reaction, a melting curve analysis was performed. Fluorescence signals of FAM, ROX, CY and HEX channels are collected in the heating process.
FIG. 1 is a graph showing the detection results of a multiplex nucleic acid detection kit for 15 sexually transmitted infectious pathogens according to an embodiment of the present invention. All the detection objects covered by the invention can detect the characteristic melting peak without any non-specific cross signal. Typical positive specimen detection results, black solid lines indicate detected pathogen DNA, and black dashed lines indicate negative controls without template.
6. T m value corresponding to detection object
The invention uses four fluorescent channels, FAM channels detect 5 pathogens, 5 medium sub-probes and two fluorescent probes, wherein three medium probes share one fluorescent probe, three different melting points are generated from low to high, and the other two medium probes share the other fluorescent probe, and two different melting points are regenerated from low to high. The ROX channel detects 5 pathogens using 6 mediator probes and two fluorescent probes, wherein three mediator probes share one fluorescent probe, two different melting points are generated from low to high, and the other three mediator probes share the other fluorescent probe, and three different melting points are generated from low to high. Cy5 channel detects 5 pathogens using 5 mediator probes and two fluorescent probes, wherein three mediator probes share one fluorescent probe, three different melting points are generated from low to high, and the other two mediator probes share the other fluorescent probe, and two different melting points are regenerated from low to high. HEX channel detects internal standard, using 1 mediator probe and one fluorescent probe. Fifteen times of T m values corresponding to the detection objects are shown in Table 4.
TABLE 4T m values for each test object
Interpretation of test results:
1) The sample should have internal control peak (IC) in HEX channel, negative control has no specific melting peak;
2) A melting peak appears at the corresponding melting point position of the corresponding channel of the specimen, and the specimen is indicated to detect the pathogen;
3) The melting points of the above tables deviate by approximately + -0.5deg.C around their values during practical experiments.
7. Clinical sample detection
The clinical specimens are detected by using 15 sexually transmitted infectious pathogen multiplex nucleic acid detection kits, 50 clinical cervical secretion samples are selected as samples to be detected, the samples are detected by using a single real-time PCR system, and the detection result and the verification method result have good consistency. The detection results of the clinical samples are shown in fig. 2, and it can be seen that the detection signals and the distinguishing effects of different pathogens are good.
Fig. 3 shows the detection results of typical positive clinical specimens, and as can be seen from fig. 3, 4 positive specimens, namely specimen a, specimen B, specimen C and specimen D, all have melting peaks at the corresponding melting point positions of the corresponding channels, and all have internal control peaks (rnarep) at the HEX channels. Sample a detected HCMV and UU in FAM channel, CT and CY5 channel detected quadruple infection of GI pathogen in ROX channel, while sample B, sample C and sample D all detected mixed infection of four different pathogens. From the sample detection results, most sexually transmitted infectious pathogens exist in the form of multiple infections, and the invention has the capability of accurately distinguishing various sexually transmitted infectious pathogens, avoids repeated screening for multiple times, and reduces the sample demand.
The invention establishes a kit for directly detecting sexually transmitted infectious pathogens, can accurately detect 15 sexually transmitted pathogens, has simple detection method, high specificity, high sensitivity and high flux, and can strengthen early diagnosis of asymptomatic patients or patients with mild symptoms. In addition, the method can be combined with and complemented with morphological detection results and biochemical function detection results, so that misdiagnosis or missed diagnosis caused by incomplete and incomplete unilateral detection results is avoided, and the method has great significance in accurate diagnosis and correct treatment of STIs.
Sequence listing
<110> Xiamen university
<120> Kit for multiplex nucleic acid detection of sexually transmitted infection
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gcaagccctc acgtagcgaa ctactcttga catccataga agaac 45
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gcaagccctc acgtagcgaa tgttcatccg ccatattgtr ttg 43
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cacacctctc gatataatcc gyccttcaac atcagtgaaa atct 44
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gcaagccctc acgtagcgaa tatctcaggt agaagggagg gct 43
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gcaagccctc acgtagcgaa gacacagcac tcgtcttcaa ctc 43
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gcaagccctc acgtagcgaa gggatcttmg gacctttcgg 40
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gcaagccctc acgtagcgaa ggcggccaat ctctcaat 38
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tgtccagcgc tgtgatatca gctagttggt ggggtaaagg c 41
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gcaagccctc acgtagcgaa accttgatgg tcagcaaaac tt 42
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gcaagccctc acgtagcgaa caatcagtag grrcattacc agt 43
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gtccacactg tcaaaggggt ttaaatggyg agcctatctt tgatc 45
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gctgcaaaaa actcaacgat gtggaagtca gcagc 35
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gcaagccctc acgtagcgaa gaccgtccta tacaagttgg at 42
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gcaagccctc acgtagcgaa ggttcaaaac gaatagcagt acc 43
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catcgttgag acttgtttga agtgaatagt gcattagtat t 41
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<400> 40
gcaagccctc acgtagcgaa gcaacaccga cttcttcggt 40
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gcaagccctc acgtagcgaa ctgccgccga aatcatagct 40
<210> 42
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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cacatcgttg agtgaagcga agaaacagaa cggcgacgg 39
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<400> 43
gcaagccctc acgtagcgaa ctcttcaagt ggaaccacta cc 42
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<212> DNA
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gacttccaca taagacggtt ggcgtctatt atggccg 37
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<400> 46
ccggcgggga gggaccgtcg tggcaggagg agcagctcac caggccgccg g 51
<210> 47
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gcaagccctc acgtagcgaa caggygcttc atgtactact aac 43
<210> 48
<211> 43
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 48
gcaagccctc acgtagcgaa ggagcatctt gtaatctttg gtc 43
<210> 49
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gccacgacgg tttagctcct attgccaacg tattgga 37
<210> 50
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<400> 50
gcaagccctc acgtagcgaa gtaaagttgc ctccggctgg 40
<210> 51
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 51
gcaagccctc acgtagcgaa ctgctatggc gtcagacgac 40
<210> 52
<211> 36
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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cctggtgagc tccgacaagg agtggctgat ctcaga 36
<210> 53
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atcgccataa aagatagacc agagagtcag agcggcgat 39
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 54
gcaagccctc acgtagcgaa gacttcagca tggcggtgtt 40
<210> 55
<211> 40
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 55
gcaagccctc acgtagcgaa gagcggctgt ctccacaagt 40
<210> 56
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 56
ctctctctgg ttctgacctg aaggctctgc gcg 33
Claims (8)
1. A kit for detecting sexually transmitted infection multiple nucleic acid is characterized by comprising a sexually transmitted pathogen detection primer and a probe, wherein the sequences of the primer and the probe are shown in SEQ ID No. 1-56.
2. The kit for detecting multiple nucleic acids of sexually transmitted infection according to claim 1, wherein the primer-probe set of the kit comprises 32 tailed primers, 16 vector probes and 7 universal probes:
the sequence of the universal primer Tag is shown as SEQ ID No. 1;
the sequence of the fluorescent probe P-U1-MB-FAM is shown as SEQ ID NO. 2;
The tailing primer for specifically amplifying the human cytomegalovirus HCMV gene sequence has an upstream primer HCMV-F sequence shown as SEQ ID NO.3 and a downstream primer HCMV-R sequence shown as SEQ ID NO. 4;
The vector probe HCMV-MP for detecting the human cytomegalovirus HCMV gene has a sequence shown in SEQ ID NO. 5;
Tail primer for specifically amplifying HSV-1 gene sequence of herpes simplex virus type 1, wherein the upstream primer HSV1-F sequence is shown as SEQ ID NO.6, and the downstream primer HSV1-R sequence is shown as SEQ ID NO. 7;
The vector probe HSV1-MP for detecting the HSV-1 gene of the herpes simplex virus 1 has a sequence shown as SEQ ID NO. 8;
Tail primer for specifically amplifying HSV-2 gene sequence of herpes simplex virus 2, wherein the upstream primer HSV2-F sequence is shown as SEQ ID NO.9, and the downstream primer HSV2-R sequence is shown as SEQ ID NO. 10;
The vector probe HSV2-MP for detecting the HSV-2 gene of the herpes simplex virus 2 has a sequence shown as SEQ ID NO. 11;
The sequence of the fluorescent probe P-U3-MB-FAM is shown as SEQ ID NO. 12;
The tail primer for specifically amplifying the trichomonas vaginalis TV gene sequence has an upstream primer TV-F sequence shown as SEQ ID NO.13 and a downstream primer TV-R sequence shown as SEQ ID NO. 14;
The vector probe TV-MP for detecting trichomonas vaginalis TV gene has a sequence shown as SEQ ID NO. 15;
the primer for adding tail of specific amplification ureaplasma urealyticum UU gene sequence, wherein the upstream primer UU-F sequence is shown as SEQ ID NO.16, and the downstream primer UU-R sequence is shown as SEQ ID NO. 17;
the vector probe UU-MP for detecting the ureaplasma urealyticum UU gene has a sequence shown as SEQ ID NO. 18;
the sequence of the fluorescent probe P-U2-MB-ROX is shown as SEQ ID NO. 19;
The primer for specifically amplifying the tailing of the HD gene sequence of the haemophilus ducreyi has an upstream primer HD-F sequence shown in SEQ ID NO.20 and a downstream primer HD-R sequence shown in SEQ ID NO. 21;
the vector probe HD-MP for detecting the Hd gene of the haemophilus ducreyi has a sequence shown in SEQ ID No. 22;
the tail primer for specifically amplifying the neisseria gonorrhoeae NG gene sequence has an upstream primer NG-F sequence shown as SEQ ID NO.23 and a downstream primer NG-R sequence shown as SEQ ID NO. 24;
The vector probe NG-MP for detecting the neisseria gonorrhoeae NG gene has a sequence shown as SEQ ID NO. 25;
The sequence of the fluorescent probe P-U1-MB-LNA-ROX is shown as SEQ ID NO. 26;
The primer for specifically amplifying the tail of the treponema pallidum TP gene sequence has an upstream primer TP-F sequence shown as SEQ ID NO.27 and a downstream primer TP-R sequence shown as SEQ ID NO. 28;
The intermediate probe TP-MP for detecting treponema pallidum TP gene has a sequence shown in SEQ ID NO. 29;
The tailing primer for specifically amplifying the Chlamydia trachomatis CT gene sequence has an upstream primer CT-F sequence shown as SEQ ID NO.30 and a downstream primer CT-R sequence shown as SEQ ID NO. 31;
The vector probe CT-MP for detecting the Chlamydia trachomatis CT gene has a sequence shown in SEQ ID NO. 32;
tail primer for specific amplification of genital mycoplasma MG gene sequence, its upstream primer MG-F sequence is shown as SEQ ID NO.33, and its downstream primer MG-R sequence is shown as SEQ ID NO. 34;
the vector probe MG-MP for detecting the mycoplasma genitalium MG gene has a sequence shown as SEQ ID NO. 35;
the sequence of the fluorescent probe P-U2-MB-CY5 is shown by SEQ ID NO. 36;
the tail primer for specifically amplifying the gene sequence of the micro ureaplasma UP has an upstream primer UP-F sequence shown as SEQ ID NO.37 and a downstream primer UP-R sequence shown as SEQ ID NO. 38;
The medium probe UP-MP for detecting the gene of the ureaplasma minutissimum has a sequence shown as SEQ ID NO. 39;
Tail primer for specific amplification of granuloma capsular bacillus GI gene sequence, its upstream primer GI-F sequence is shown in SEQ ID NO.40, and its downstream primer GI-R sequence is shown in SEQ ID NO. 41;
The vector probe GI-MP for detecting the GI gene of the granulomatous capsular bacillus has a sequence shown as SEQ ID NO. 42;
The tail primer for specifically amplifying the herpes zoster virus VZV gene sequence has an upstream primer VZV-F sequence shown as SEQ ID NO.43 and a downstream primer VZV-R sequence shown as SEQ ID NO. 44;
the vector probe VZV-MP for detecting the herpes zoster virus VZV gene has a sequence shown as SEQ ID NO. 45;
the sequence of the fluorescent probe P-U3-MB-CY5 is shown by SEQ ID NO. 46;
The primer for specifically amplifying the tailing of the human mycoplasma MH gene sequence has an upstream primer MH-F sequence shown as SEQ ID NO.47 and a downstream primer MH-R sequence shown as SEQ ID NO. 48;
the base sequence of the vector probe MH-MP used for detecting the human mycoplasma MH gene is shown as SEQ ID NO. 49;
The tailing primer for specifically amplifying the human herpesvirus 8-type HHV-8 gene sequence has the upstream primer HHV8-F sequence shown as SEQ ID NO.50 and the downstream primer HHV8-R sequence shown as SEQ ID NO. 51;
the vector probe HHV8-MP for detecting the human herpesvirus 8-type HHV-8 gene has the sequence shown in SEQ ID NO. 52;
The sequence of the fluorescent probe P-U1-MB-HEX is shown as SEQ ID NO. 53;
The tailing primer for specifically amplifying the humanized RPP30 gene sequence has an upstream primer RPP30-F sequence shown as SEQ ID NO.54 and a downstream primer RPP30-R sequence shown as SEQ ID NO. 55;
The vector probe RPP30-MP for detecting the human RPP30 gene has a sequence shown in SEQ ID NO. 56.
3. The kit for detecting multiple nucleic acids for sexually transmitted infection according to claim 2, wherein the concentration ratio of the upstream primer, the downstream primer and the mediator probe in each primer probe set is 1:1:10.
4. A kit for multiplex nucleic acid detection for sexually transmitted infections according to claim 1, characterized in that said kit employs a single tube four-channel for simultaneous detection of 15 sexually transmitted infectious pathogens, said 15 sexually transmitted infectious pathogens comprising bacteria, viruses, chlamydia, mycoplasma, parasites.
5. The kit for detecting multiple nucleic acids of sexually transmitted infection according to claim 1, further comprising a PCR reaction solution.
6. A kit for the detection of sexually transmitted infection (STRIM) multiple nucleic acids according to claim 5, wherein the PCR reaction solution comprises PCR buffer, dNTPs, mgCl 2, SSB, taq01 enzyme and UNG enzyme.
7. The kit for detecting multiple nucleic acids of sexually transmitted infection according to claim 1, further comprising a positive quality control and a negative quality control.
8. The kit for detecting multiple nucleic acids of sexually transmitted infection according to claim 7, wherein said positive quality control is a cloning plasmid and said negative quality control is sterilized purified water.
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| CN112831581A (en) * | 2021-02-10 | 2021-05-25 | 华东医院 | Detection system for pathogen capable of curing sexually transmitted infection, kit and application thereof |
| CN115449549B (en) * | 2022-10-27 | 2023-12-26 | 上海捷易生物科技有限公司 | Kit for screening neonatal immunodeficiency disease and preparation method thereof |
| CN118166135A (en) * | 2024-05-11 | 2024-06-11 | 深圳闪量科技有限公司 | Primer pool for simultaneously detecting multiple pathogens of genital tract infection and PCR rapid detection kit |
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| CN106868213A (en) * | 2017-03-21 | 2017-06-20 | 重庆迪安医学检验中心有限公司 | A kind of primer and its application for detecting parainfluenza virus and coronavirus simultaneously based on melting curve method single tube |
| CN107630098A (en) * | 2017-11-13 | 2018-01-26 | 湖南圣湘生物科技有限公司 | Fluorescent PCR detection architecture, kit and detection method for joint-detection various respiratory road bacterium |
| KR20190124059A (en) * | 2018-04-25 | 2019-11-04 | 주식회사 이원생명과학연구원 | Kits for Detecting Pathogens of Sexually Transmitted Infections |
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| US10722562B2 (en) * | 2008-07-23 | 2020-07-28 | Immudex Aps | Combinatorial analysis and repair |
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| CN106868213A (en) * | 2017-03-21 | 2017-06-20 | 重庆迪安医学检验中心有限公司 | A kind of primer and its application for detecting parainfluenza virus and coronavirus simultaneously based on melting curve method single tube |
| CN107630098A (en) * | 2017-11-13 | 2018-01-26 | 湖南圣湘生物科技有限公司 | Fluorescent PCR detection architecture, kit and detection method for joint-detection various respiratory road bacterium |
| KR20190124059A (en) * | 2018-04-25 | 2019-11-04 | 주식회사 이원생명과학연구원 | Kits for Detecting Pathogens of Sexually Transmitted Infections |
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