CN110656189A - Fluorescent PCR kit for single-tube multiple rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum - Google Patents
Fluorescent PCR kit for single-tube multiple rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum Download PDFInfo
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Abstract
The invention relates to the technical field of pathogen detection, in particular to a fluorescent PCR kit for single-tube multiple rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum. The kit comprises PCR reaction liquid, a PCR starter, a positive quality control product and a negative quality control product; the PCR reaction solution comprises a PCR accelerator, a PCR buffer, a primer and a probe for detecting chlamydia trachomatis, a primer and a probe for detecting gonococcus, a primer and a probe for detecting ureaplasma urealyticumA probe, a primer and a probe for detecting human GAPDH gene DNA, DNA polymerase, UNG enzyme, dNTP and a preservative; the PCR accelerator consists of DMSO, Tween20 and glycerol; PCR buffers included tris (hydroxymethyl) methylglycine and potassium acetate; PCR initiator includes Mn (OAc)2、MgCl2And a preservative. The kit provided by the invention realizes the technical effects of high sensitivity, strong specificity, simple and convenient operation and shorter time consumption.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a fluorescent PCR kit for single-tube multiple rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum.
Background
STD (sexually transmitted diseases) occurs in genital areas, including syphilis, gonorrhea, chancroid, lymphogranuloma veneris, and granuloma inguinale. Sexually transmitted diseases are a group of common infectious diseases widely prevalent in the world, and show the trends of expanding epidemic scope, reducing the onset age and increasing drug-resistant strains, particularly the great increase of AIDS, which becomes a serious public health problem. The prevention and treatment of venereal diseases is a very difficult and long-term task. Common pathogens include viruses, chlamydia, mycoplasma, spirochetes, bacteria, fungi, protozoa, and parasites, of which Chlamydia Trachomatis (CT), gonococcus (NG), and Ureaplasma Urealyticum (UU) are common pathogens.
Multiplex PCR (multiplex PCR) is an improved PCR technique based on general PCR, in which multiple pairs of specific primers are added to a PCR reaction system to amplify multiple target fragments for multiple DNA templates or different regions of the same template. This concept was introduced by Chamberian et al, prior to 1988. Since multiplex PCR simultaneously amplifies a plurality of target genes, it has the advantages of saving time, reducing cost and improving efficiency, especially saving precious experimental samples, once it is proposed, it is popular among many researchers, and it is rapidly developed, and multiplex PCR has become a mature and important research means in various fields of life science. Because multiple PCR requires specific amplification of multiple sites in the same reaction system, the technical difficulty is increased, an ideal multiple PCR reaction system is not simple mixing of single PCR, and comprehensive analysis and repeated tests are required to be carried out on target products to establish a proper reaction system and reaction conditions. The technical elements of the multiplex PCR mainly include target fragment selection, primer design, renaturation temperature and time, extension temperature and time, the dosage of each reaction component and the like.
At present, the harbor dragon organism (patent number 200910164977.6), the Jiangsu Master organism (patent number 201310275243.1) and the Suzhou English innovative organism (patent number 201610755715.7) have related patents about CT/NG/UU single-tube triple fluorescence PCR detection, and the aims are to overcome the defects of poor specificity, low sensitivity, long detection time and the like of CT/NG/UU detection.
However, the existing multiplex PCR technology still has the defects of poor detection specificity, low sensitivity and the like, and the multiplex PCR reagent is difficult to realize rapid detection because of simultaneously detecting a plurality of target sequences, and the reagent is still stored by low-temperature freezing. In addition, the amplification reagents use exogenous plasmids as internal standards, and only the process of extraction and amplification is monitored.
Disclosure of Invention
In view of this, the invention provides a fluorescent PCR kit for single-tube multiplex rapid detection of Chlamydia trachomatis, gonococcus and ureaplasma urealyticum. The fluorescent PCR kit realizes the technical effects of high sensitivity, strong specificity, simple and convenient operation and shorter time consumption.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a PCR accelerator for detecting chlamydia trachomatis, gonococcus and ureaplasma urealyticum, which consists of DMSO, Tween20 and glycerol.
Preferably, in the PCR accelerator, the volume ratio of DMSO, Tween20 to glycerol is (1-20): (0.01-0.15): (1-20).
Preferably, in the PCR accelerator, the volume ratio of DMSO, Tween20 to glycerol is (6-10): (0.02-0.08): (1-3).
More preferably, the volume ratio of DMSO, Tween20 and glycerol in the PCR accelerator is 8: 0.05: 2.
the invention also provides a fluorescent PCR kit for single-tube multiple rapid detection of Chlamydia trachomatis, gonococcus and ureaplasma urealyticum, wherein the fluorescent PCR kit comprises PCR reaction liquid, a PCR starter, a positive quality control product and a negative quality control product;
the PCR reaction solution comprises a PCR accelerator, a PCR buffer, a primer and a probe for detecting chlamydia trachomatis, a primer and a probe for detecting gonococcus, a primer and a probe for detecting ureaplasma urealyticum, a primer and a probe for detecting human GAPDH gene DNA, DNA polymerase, UNG enzyme, dNTP and a preservative;
the PCR accelerator consists of DMSO, Tween20 and glycerol;
PCR buffers included tris (hydroxymethyl) methylglycine and potassium acetate;
PCR initiator includes Mn (OAc)2、MgCl2And a preservative.
Preferably, the DNA polymerase is rTth DNA polymerase.
In the present invention, dNTP is a combination of dATP, dGTP, dCTP and dUTP.
Preferably, the primer for detecting chlamydia trachomatis consists of SEQ ID No: 1 and the forward primer shown in SEQ ID No: 2 is shown in the specification;
the primer for detecting the gonococcus consists of SEQ ID No: 3 and the forward primer shown in SEQ ID No: 4, and a reverse primer composition;
the primer for detecting ureaplasma urealyticum consists of SEQ ID No: 5 and SEQ ID No: 6, reverse primer composition;
primers for detecting human GAPDH gene DNA consist of SEQ ID No: 7 and SEQ ID No: 8 is shown in the specification;
the probe sequence of the chlamydia trachomatis is shown as SEQ ID No: 9 is shown in the figure;
the probe sequence of the gonococcus is shown as SEQ ID No: 10 is shown in the figure;
the probe sequence of ureaplasma urealyticum is shown as SEQ ID No: 11 is shown in the figure;
the probe sequence of human GAPDH gene DNA is shown in SEQ ID No: shown at 12.
SEQ ID No：1：5'-TGGATTACCTATAACTGTAGACTCGGC-3'
SEQ ID No：2：5'-GATAAAGCATCATGCAACATTAACC-3'
SEQ ID No：3：5'-GAAGCGGTAGGAAGCAGTGGC-3'
SEQ ID No：4：5'-GCTGGTATGTTCGCGTTTCTGAC-3'
SEQ ID No：5：5'-GTTGCAATTACATCAGCTGAAAATAAAAC-3'
SEQ ID No：6：5'-TAGTTAATTGTTCTTTAGCTTTTGGTTCAC-3'
SEQ ID No：7：5'-ACAGTCCATGCCATCACTG-3'
SEQ ID No：8：5'-CTGTGGCGTGATGGCCGT-3'
SEQ ID No：9：FAM-GAAGAGCTTTTGCGGCGTCGTATCA-BHQ1；
SEQ ID No：10：ROX-CGCAAGCTGTTTCAGCTTTGGTACCC-BHQ2；
SEQ ID No：11：Cy5-AAAACGCAACAACAAAAGGTCACTTACTTAA CAA-BHQ2；
SEQ ID No：12：HEX-CTCAGAAGACTGTGGATGGCC-BHQ1。
Preferably, the concentrations of the respective components in the PCR reaction solution are as follows:
1 v/v% -20 v/v% DMSO, 0.01 v/v% -0.15 v/v% Tween20, and 1 v/v% -20 v/v% glycerol;
10-100 mM tris (hydroxymethyl) methylglycine, 50-150 mM potassium acetate;
0.1-0.5 mu M of chlamydia trachomatis forward primer and 0.1-0.5 mu M of chlamydia trachomatis reverse primer;
0.1-0.5 mu M gonococcus forward primer and 0.1-0.5 mu M gonococcus reverse primer;
0.1-0.5 mu M of ureaplasma urealyticum forward primer and 0.1-0.5 mu M of ureaplasma urealyticum reverse primer;
0.1-0.5 mu M human GAPDH gene DNA forward primer and 0.1-0.5 mu M human GAPDH gene D NA reverse primer;
0.05-0.2 mu M Chlamydia trachomatis probe, 0.05-0.2 mu M gonococcus probe, 0.05-0.2 mu M ureaplasma urealyticum probe, 0.01-0.1 mu M human GAPDH gene DNA probe;
5 to 15U of DNA polymerase, 0.5 to 2U of UNG enzyme, 0.1 to 0.3mM of dATP, 0.1 to 0.3mM of dGTP, 0.1 to 0.3mM of dCTP, 0.2 to 0.6mM of dUTP, and 0.05 to 0.2 w/t% of preservative.
Preferably, the concentrations of the components in the PCR reaction solution are as follows:
8 v/v% DMSO, 0.05 v/v% Tween20, 2 v/v% glycerol;
50mM tris (hydroxymethyl) methylglycine (pH8.3), 100mM potassium acetate;
0.3 mu M Chlamydia trachomatis forward primer, 0.3 mu M Chlamydia trachomatis reverse primer;
0.3 mu M gonococcus forward primer and 0.3 mu M gonococcus reverse primer;
0.3 mu M of ureaplasma urealyticum forward primer and 0.3 mu M of ureaplasma urealyticum reverse primer;
0.3. mu.M human GAPDH gene DNA forward primer, 0.15. mu.M human GAPDH gene DNA reverse primer;
0.1. mu.M Chlamydia trachomatis probe, 0.1. mu.M gonococcus probe, 0.1. mu.M ureaplasma urealyticum probe, 0.05. mu.M human GAPDH gene DNA probe;
10U DNA polymerase, 1U UNG enzyme, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.4mM dUTP, 0.1 w/t% preservative.
Preferably, tris (hydroxymethyl) methylglycine has a pH of 8.3.
Preferably, the concentrations of the components in the PCR promoter are as follows: 1 to 5mM Mn (OAc)2、0.5～1.5mM MgCl20.05-0.2 w/t% preservative.
Preferably, the concentrations of the components in the PCR promoter are: 3mM Mn (OAc)2、1mM MgCl20.1 w/t% preservative.
Preferably, the volume ratio of the PCR reaction solution to the PCR starter is (10-30): (5-15).
Preferably, the volume ratio of the PCR reaction solution to the PCR initiator is 2: 1.
preferably, the preservative is sodium azide.
In the invention, the negative quality control product is a swab rinsing lotion which does not contain chlamydia trachomatis, gonococcus and ureaplasma urealyticum;
the positive quality control product comprises: plasmids containing fragments of Chlamydia trachomatis, plasmids containing fragments of gonococci, and plasmids containing fragments of ureaplasma urealyticum.
In the invention, the human endogenous GAPDH gene is used as an internal control, and the sampling process is monitored to avoid false negative. In addition, human endogenous genes such as beta-actin (beta actin) gene, beta 2-MG (beta 2-microglobulin), PBGD (depigmenting element deaminase) and the like are also within the scope of the present invention.
The invention provides a single-tube multiplex rapid fluorescence PCR kit for detecting chlamydia trachomatis, gonococcus and ureaplasma urealyticum, which comprises PCR reaction liquid, a PCR starter, a positive quality control product and a negative quality control product; the PCR reaction solution comprises a PCR accelerator, a PCR buffer, a primer and a probe for detecting chlamydia trachomatis, a primer and a probe for detecting gonococcus, a primer and a probe for detecting ureaplasma urealyticum, a primer and a probe for detecting human GAPDH gene DNA, DNA polymerase, UNG enzyme, dNTP and a preservative; the PCR accelerator consists of DMSO, Tween20 and glycerol; PCR buffers included tris (hydroxymethyl) methylglycine and potassium acetate; PCR initiator includes Mn (OAc)2、MgCl2And a preservative. The invention has the technical effects that:
the fluorescent PCR kit disclosed by the invention realizes the technical effects of high sensitivity, strong specificity, simplicity and convenience in operation and shorter time consumption;
meanwhile, the types of the samples detected by the method are genital tract swabs, and chlamydia trachomatis, gonococcus and ureaplasma urealyticum are all pathogenic microorganisms parasitizing in human cells, and false negative is easy to occur if epithelial cells of the human genital tract are not collected by swab sampling;
the invention realizes rapid detection mainly by the PCR rapid detection technology and the PCR reagent preservation technology at 2-8 ℃, thereby not only saving PCR detection time, but also saving operation time in the use process;
the harbor dragon organism and the Jiangsu major organism respectively design 3 pairs of primers and 3 specific probes aiming at CT/NG/UU, the harbor dragon organism adopts a Taqman MGB probe, the cost is high, the major organism adopts the Taqman probe, the CT/NG/UU is detected in parallel in the same tube, the Suzhou English new creature adopts a molecular beacon probe, an internal standard monitoring system is added, the CT/NG/UU is detected simultaneously in the same reaction system, the effectiveness of PCR reaction and the effectiveness of experimental results can be monitored in real time, false negative and false positive are avoided, however, the false negative caused by improper sample collection cannot be effectively monitored due to the fact that the internal standard of the molecular beacon is artificially added plasmid, and the defects of the prior art, such as long detection time and low detection sensitivity, exist due to the defects of the multiple PCR technology. The invention adopts a Taqman probe method, has lower cost than a Taqman MGB probe, is a quadruple PCR system with an internal control compared with a triple PCR system of major organisms, and has a monitoring function on whether sampling is effective or not besides monitoring extraction and amplification compared with the method that a plasmid is used as an exogenous internal standard for monitoring extraction and amplification newly created in English in Suzhou. The kit is stored for at least 1 year at the temperature of 2-8 ℃, and is more convenient and faster to use. In addition, the invention can complete detection within 50 minutes, and meets the requirement of clinical rapid detection.
The PCR accelerator provided by the invention not only reduces mismatching in the multiplex PCR amplification process, improves the efficiency and specificity of multiplex PCR amplification, and improves the annealing and extension rate of the primer and the template, thereby shortening the annealing and extension time in the amplification cycle, completing the fluorescent PCR detection of 45 amplification cycles within 50 minutes, and greatly shortening the detection time. Meanwhile, the PCR product can be used as an enzyme stabilizer, so that the storage stability of the DNA polymerase is improved, and the effect of stably storing the PCR reaction solution for at least 1 year at 2-8 ℃ is realized.
Drawings
FIG. 1 is a comparison of different accelerator protocols in the detection of Chlamydia trachomatis DNA;
FIG. 2 is a comparison of different accelerator protocols in gonococcal DNA assays;
FIG. 3 is a comparison of different accelerator protocols in the DNA detection of ureaplasma urealyticum.
Detailed Description
The invention discloses a single-tube multiple-rapid fluorescence PCR kit for detecting chlamydia trachomatis, gonococcus and ureaplasma urealyticum, and a person skilled in the art can use the contents to reference the text and appropriately improve the process parameters to realize the detection. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Interpretation of terms:
the PCR rapid detection technology comprises the following steps: the time required to complete a PCR reaction on a PCR instrument depends on several factors: 1. module temperature rise and fall time, 2, module and PCR intraductal temperature balance time, 3, extension time, 4, cycle number. Among them, factors 1 and 2 are generally implemented by a rapid PCR instrument, but there are disadvantages in that temperature overshoot due to rapid temperature change and the actual temperature in the PCR tube is far from the set temperature, so that rapid PCR generally requires the following measures to balance the reaction temperature and time: firstly, the temperature of the module is overshot and is reduced to the target temperature when the temperature in the tube is quickly reached, but the actual temperature overshot can be caused by too high overshooting, and the PCR reaction result is influenced; secondly, the volume of the PCR reaction solution is reduced, but the reliability of the PCR reaction is reduced when the volume is reduced; thirdly, the thickness of the PCR tube is reduced, but the strength is not enough when the PCR tube is too thin; fourthly, the attaching degree of the PCR tube and the module is increased. Factor 3 extension time is related to the length of the target sequence and the synthesis rate of the DNA polymerase and can be achieved by using high-speed DNA polymerase, but not all PCR assays can use high-speed DNA polymerase due to cost issues. The rapid PCR technology is to integrate the rapid PCR instrument with the ultra-thin tube and the high-speed DNA polymerase, etc., so as to complete the PCR reaction in a short time.
The PCR reagent storage technology at 2-8 ℃ comprises the following steps: the PCR reagent generally consists of DNA polymerase, Buffer, primers/probes, Mg and dNTP, the enzyme generally needs to be preserved at the temperature of-20 ℃, and the Buffer, the primers/probes, the Mg and the dNTP can be preserved at the temperature of 2-8 ℃, so that the common PCR reagents on the market are preserved at the temperature of-20 ℃, which is very inconvenient to use, and need to be frozen and thawed or even repeatedly frozen and thawed for the reagents, thereby not only affecting the detection speed, but also affecting the performance of the reagents. The active site of the DNA polymerase is sealed through certain modification to form the hot-start DNA polymerase, and the activity period of the DNA polymerase can be prolonged at a certain degree when the DNA polymerase is stored at 2-8 ℃, but the current PCR reagents prepared by using the hot-start DNA polymerase are stored at-20 ℃ to ensure the activity of the enzyme. The nucleic acid detection kit (COBAS TaqMan Assays) of Roche adopts double-functional DNA polymerase (Z05 DNA polymerase, a hot-start DNA polymerase with both reverse transcription function and DNA polymerase function), and amplification reagents thereof are stored at 2-8 ℃, but research and research have shown that the single Z05 DNA polymerase is also stored at-15 to-25 ℃, so that the 2-8 ℃ storage of the PCR reagent depends on the hot-start enzyme and the Buffer of the reagent to maximally protect the stability of the enzyme, and the PCR reagent needs to be realized by using PCR accelerators, such as gelatin, BSA, Tween20, trehalose and the like, so as to improve the stability of the DNA polymerase.
The reagents or instruments used in the single-tube multiple rapid fluorescence PCR kit for detecting chlamydia trachomatis, gonococcus and ureaplasma urealyticum can be purchased from the market.
The invention is further illustrated by the following examples:
example 1: kit composition
(1)20 μ L of PCR reaction:
8% DMSO, 0.05% Tween20, 2% glycerol;
50mM tris (hydroxymethyl) methylglycine (pH8.3), 100mM potassium acetate;
0.3 mu M Chlamydia trachomatis primer 1, 0.3 mu M Chlamydia trachomatis primer 2;
0.3 mu M gonococcus primer 1, 0.3 mu M gonococcus primer 2;
0.3 mu M ureaplasma urealyticum primer 1 and 0.3 mu M ureaplasma urealyticum primer 2;
0.3. mu.M human GAPDH gene DNA primer 1, 0.15. mu.M human GAPDH gene DNA primer 2;
0.1. mu.M Chlamydia trachomatis probe, 0.1. mu.M gonococcus probe, 0.1. mu.M ureaplasma urealyticum probe, 0.05. mu.M human GAPDH gene DNA probe;
10U rTth DNA polymerase, 1U UNG enzyme, 0.2 mdATP, 0.2mMdGTP, 0.2 mMdCTP, 0.4mMdUTP, 0.1% sodium azide;
(2)10 μ L PCR initiator: 3mM Mn (OAc)2、1mM MgCl20.1% sodium azide.
(3) Negative quality control product: is a swab rinsing lotion which does not contain chlamydia trachomatis, gonococcus and ureaplasma urealyticum.
(4) Strong positive quality control product: comprises a plasmid containing a segment of Chlamydia trachomatis, a plasmid containing a segment of Neurococcales, and a plasmid containing a segment of ureaplasma urealyticum.
The primer and probe sequences are as follows:
specific primers for detecting chlamydia trachomatis comprise:
chlamydia trachomatis primer 1(SEQ ID No. 1): 5'-TGGATTACCTATAACTGTAGACTCGGC-3'
Chlamydia trachomatis primer 2(SEQ ID No. 2): 5'-GATAAAGCATCATGCAACATTAACC-3'
Secondly, the specific primer for detecting the gonococcus comprises the following components:
gonococcal primer 1(SEQ ID No. 3): 5'-GAAGCGGTAGGAAGCAGTGGC-3'
Gonococcal primer 2(SEQ ID No. 4): 5'-GCTGGTATGTTCGCGTTTCTGAC-3'
③ the specific primer for detecting ureaplasma urealyticum, which comprises the following components:
ureaplasma urealyticum primer 1(SEQ ID No. 5): 5'-GTTGCAATTACATCAGCTGAAAATAAAAC-3'
Ureaplasma urealyticum primer 2(SEQ ID No. 6): 5'-TAGTTAATTGTTCTTTAGCTTTTGGTTCAC-3'
Specific primers for detecting human GAPDH gene DNA comprise:
human GAPDH gene DNA primer 1(SEQ ID No. 7): 5'-ACAGTCCATGCCATCACTG-3'
Human GAPDH gene DNA primer 2(SEQ ID No. 8): 5'-CTGTGGCGTGATGGCCGT-3'
Fifthly, the specific probe of the chlamydia trachomatis comprises:
chlamydia trachomatis probe (SEQ ID No. 9): FAM-GAAGAGCTTTTGCGGCGTCGTATCA-BHQ 1;
sixthly, the specific probe of the gonococcus comprises:
gonococcal probe (SEQ ID No. 10): ROX-CGCAAGCTGTTTCAGCTTTGGTACCC-BHQ 2;
seventhly, a specific probe of ureaplasma urealyticum, comprising:
ureaplasma urealyticum probe (SEQ ID No. 11): cy5-AAAACGCAACAACAAAAGGTCACTTACTTAACAA-BHQ 2;
specific probe of human GAPDH gene DNA, including:
human GAPDH gene DNA probe (SEQ ID No. 12): HEX-CTCAGAAGACTGTGGATGGCC-BHQ 1.
Example 2: extraction of Chlamydia trachomatis, gonococcus and ureaplasma urealyticum DNA and human GAPDH gene DNA
This example extracts and purifies Chlamydia trachomatis, Neisseria gonorrhoeae and Urea ureaplasma urealyticum DNA and human GAPDH gene DNA in a sample, wherein the sample is a genital tract swab, and a nucleic acid extraction or purification reagent of Zhengzhou Antu bioengineering GmbH is used, and the specific operation is performed according to the instruction.
Example 3: comparison of the effects of different combinations of accelerators on the DNA amplification of Chlamydia trachomatis, gonococci and ureaplasma urealyticum
Diluting the chlamydia trachomatis plasmid, the gonococcus plasmid and the ureaplasma urealyticum plasmid to 400copies/mL by using a swab rinsing washing solution, extracting and purifying nucleic acid by using a nucleic acid extraction or purification reagent of Zheng Zhou Antu bioengineering GmbH, detecting by using the kit, combining different accelerators according to table 1 to prepare a PCR reaction solution, and comparing the amplification effects of the chlamydia trachomatis, the gonococcus and the ureaplasma urealyticum DNA at a low concentration level, as shown in figures 1-3, the amplification effect of the scheme 7 is superior to that of the scheme 8, is superior to that of the combination of two accelerators, and is superior to that of a single accelerator.
TABLE 1 different Accelerator combination schemes
Example 4: minimum detection limit of the kit
The chlamydia trachomatis plasmid, gonococcus plasmid and ureaplasma urealyticum plasmid are diluted to 400, 200, 100, 50 and 25copies/mL by a swab rinsing washing solution, nucleic acid extraction and purification are carried out by adopting a nucleic acid extraction or purification reagent of Zhengzhou Anchai bioengineering GmbH, the kit is used for detection, each concentration sample is repeated for 60 times, the detection rate of each concentration sample is calculated, and thus the lowest detection limit is determined (the lowest concentration with the detection rate being not less than 95% is determined as the lowest detection limit), and the results are shown in tables 2 to 4. As is clear from the results, the minimum detection limit for Chlamydia trachomatis was 200copies/mL, the minimum detection limit for gonococci was 200copies/mL, and the minimum detection limit for ureaplasma urealyticum was 200 copies/mL.
TABLE 2 minimum detection limits for Chlamydia trachomatis detection
TABLE 3 minimum detection limits for gonococci
TABLE 4 minimum detection limits for ureaplasma urealyticum
Example 5 comparative test
Different PCR reaction solutions were prepared according to the PCR accelerator protocol in the prior patents (201811233741.9, 201711332336.8) (scheme 1, scheme 2) and compared to the study (scheme 3): diluting the chlamydia trachomatis plasmid, the gonococcus plasmid and the ureaplasma urealyticum plasmid to the lowest detection limit concentration (200copies/mL) by using a swab rinsing washing solution, extracting and purifying nucleic acid by using nucleic acid extracting or purifying reagents of Zhengzhou Antu bioengineering GmbH, and detecting by using PCR reaction solutions prepared by different PCR accelerator schemes, wherein the detection is repeated for 21 times, and the results are as follows:
TABLE 5 collage DNA detection comparison
TABLE 6 gonococcal DNA detection comparison
TABLE 7 Urea ureaplasma urealyticum DNA detection comparison
The comparison result shows that when the lowest detection limit concentration samples (200copies/mL) of the chlamydia trachomatis, the gonococcus and the ureaplasma urealyticum are detected, the research scheme can completely detect the samples, the detection rate is 100 percent, and the repeatability of the Ct value is superior to that of the existing patent schemes (scheme 1 and scheme 2).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Zhengzhou Antu bioengineering GmbH
<120> fluorescent PCR kit for single-tube multiple rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum
<130> MP1915107
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tggattacct ataactgtag actcggc 27
<210> 2
<211> 25
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gataaagcat catgcaacat taacc 25
<210> 3
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaagcggtag gaagcagtgg c 21
<210> 4
<211> 23
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gctggtatgt tcgcgtttct gac 23
<210> 5
<211> 29
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gttgcaatta catcagctga aaataaaac 29
<210> 6
<211> 30
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tagttaattg ttctttagct tttggttcac 30
<210> 7
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acagtccatg ccatcactg 19
<210> 8
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctgtggcgtg atggccgt 18
<210> 9
<211> 25
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gaagagcttt tgcggcgtcg tatca 25
<210> 10
<211> 26
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgcaagctgt ttcagctttg gtaccc 26
<210> 11
<211> 34
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aaaacgcaac aacaaaaggt cacttactta acaa 34
<210> 12
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ctcagaagac tgtggatggc c 21
Claims (10)
1. A PCR accelerator for detecting Chlamydia trachomatis, Neisseria gonorrhoeae and ureaplasma urealyticum, wherein the PCR accelerator consists of DMSO, Tween20 and glycerol.
2. The PCR accelerator according to claim 1, wherein the volume ratio of DMSO, Tween20 and glycerol is (1-20): (0.01-0.15): (1-20).
3. The PCR accelerator according to claim 2, wherein the volume ratio of DMSO, Tween20 and glycerol is (6-10): (0.02-0.08): (1-3).
4. A single-tube multiplex fluorescence PCR kit for rapidly detecting chlamydia trachomatis, gonococcus and ureaplasma urealyticum is characterized in that the fluorescence PCR kit comprises PCR reaction liquid, a PCR starter, a positive quality control product and a negative quality control product;
the PCR reaction solution comprises a PCR accelerator, a PCR buffer, a primer and a probe for detecting chlamydia trachomatis, a primer and a probe for detecting gonococcus, a primer and a probe for detecting ureaplasma urealyticum, a primer and a probe for detecting human GAPDH gene DNA, DNA polymerase, UNG enzyme, dNTP and a preservative;
the PCR accelerator consists of DMSO, Tween20 and glycerol;
the PCR buffer comprises tris (hydroxymethyl) methylglycine and potassium acetate;
the PCR promoter comprisesMn(OAc)2、MgCl2And a preservative.
5. The fluorescent PCR kit of claim 1, wherein the DNA polymerase is rTth DNA polymerase and the dNTPs are a combination of dATP, dGTP, dCTP, dUTP.
6. The fluorescent PCR kit of any one of claims 1 to 5, wherein the primer for detecting Chlamydia trachomatis consists of SEQ ID No: 1 and the forward primer shown in SEQ ID No: 2 is shown in the specification;
the primer for detecting the gonococcus consists of SEQ ID No: 3 and the forward primer shown in SEQ ID No: 4, and a reverse primer composition;
the primer for detecting ureaplasma urealyticum consists of SEQ ID No: 5 and SEQ ID No: 6, the reverse primer;
the primer for detecting human GAPDH gene DNA consists of SEQ ID No: 7 and SEQ ID No: 8 is shown in the specification;
the probe sequence of the chlamydia trachomatis is shown as SEQ ID No: 9 is shown in the figure;
the probe sequence of the gonococcus is shown as SEQ ID No: 10 is shown in the figure;
the probe sequence of the ureaplasma urealyticum is shown as SEQ ID No: 11 is shown in the figure;
the probe sequence of the human GAPDH gene DNA is shown in SEQ ID No: shown at 12.
7. The fluorescent PCR kit according to claim 6, wherein the concentrations of the respective components in the PCR reaction solution are as follows:
1 v/v% -20 v/v% DMSO, 0.01 v/v% -0.15 v/v% Tween20, and 1 v/v% -20 v/v% glycerol;
10-100 mM tris (hydroxymethyl) methylglycine, 50-150 mM potassium acetate;
0.1-0.5 mu M of chlamydia trachomatis forward primer and 0.1-0.5 mu M of chlamydia trachomatis reverse primer;
0.1-0.5 mu M gonococcus forward primer and 0.1-0.5 mu M gonococcus reverse primer;
0.1-0.5 mu M of ureaplasma urealyticum forward primer and 0.1-0.5 mu M of ureaplasma urealyticum reverse primer;
0.1-0.5 mu M human GAPDH gene DNA forward primer and 0.1-0.5 mu M human GAPDH gene DNA reverse primer;
0.05-0.2 mu M Chlamydia trachomatis probe, 0.05-0.2 mu M gonococcus probe, 0.05-0.2 mu M ureaplasma urealyticum probe, 0.01-0.1 mu M human GAPDH gene DNA probe;
5 to 15U of DNA polymerase, 0.5 to 2U of UNG enzyme, 0.1 to 0.3mM of dATP, 0.1 to 0.3mM of dGTP, 0.1 to 0.3mM of dCTP, 0.2 to 0.6mM of dUTP, and 0.05 to 0.2 w/t% of preservative.
8. The fluorescent PCR kit of claim 7, wherein the concentration of each component in the PCR promoter is: 1 to 5mM Mn (OAc)2、0.5～1.5mM MgCl20.05-0.2 w/t% preservative.
9. The fluorescent PCR kit according to claim 1, wherein the volume ratio of the PCR reaction solution to the PCR initiator is (10-30): (5-15).
10. The fluorescent PCR kit of claim 1, wherein the negative quality control material is a swab rinse solution free of Chlamydia trachomatis, gonococci, and ureaplasma urealyticum;
the positive quality control product comprises: plasmids containing fragments of Chlamydia trachomatis, plasmids containing fragments of gonococci, and plasmids containing fragments of ureaplasma urealyticum.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113444821A (en) * | 2021-07-01 | 2021-09-28 | 江苏汇先医药技术有限公司 | Kit and method for synchronously detecting multiple genital tract pathogens |
| CN114164288A (en) * | 2021-12-24 | 2022-03-11 | 苏州中科先进技术研究院有限公司 | Primer probe composition, kit and method for detecting ureaplasma urealyticum |
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| CN101497926A (en) * | 2008-12-23 | 2009-08-05 | 武汉大学 | Reagent kit for rapidly and synchronously detecting Chlamydi trachomatis, Urealpasma parvum and Ureaplasma urealyticum |
| CN103409508A (en) * | 2013-07-02 | 2013-11-27 | 江苏硕世生物科技有限公司 | Gonococcus/ureaplasma urealyticum/chlamydia trachomatis triple nucleic acid detection kit |
| CN107937580A (en) * | 2017-12-26 | 2018-04-20 | 湖南圣湘生物科技有限公司 | The application method of the primer and probe of urogenital tract microorganism detection, kit and kit |
| CN108977580A (en) * | 2018-08-13 | 2018-12-11 | 郑州安图生物工程股份有限公司 | A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation |
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| CN101497926A (en) * | 2008-12-23 | 2009-08-05 | 武汉大学 | Reagent kit for rapidly and synchronously detecting Chlamydi trachomatis, Urealpasma parvum and Ureaplasma urealyticum |
| CN103409508A (en) * | 2013-07-02 | 2013-11-27 | 江苏硕世生物科技有限公司 | Gonococcus/ureaplasma urealyticum/chlamydia trachomatis triple nucleic acid detection kit |
| CN107937580A (en) * | 2017-12-26 | 2018-04-20 | 湖南圣湘生物科技有限公司 | The application method of the primer and probe of urogenital tract microorganism detection, kit and kit |
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Cited By (3)
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| CN113444821A (en) * | 2021-07-01 | 2021-09-28 | 江苏汇先医药技术有限公司 | Kit and method for synchronously detecting multiple genital tract pathogens |
| CN113444821B (en) * | 2021-07-01 | 2023-12-19 | 江苏汇先医药技术有限公司 | Kit and method for synchronously detecting various genital tract pathogens |
| CN114164288A (en) * | 2021-12-24 | 2022-03-11 | 苏州中科先进技术研究院有限公司 | Primer probe composition, kit and method for detecting ureaplasma urealyticum |
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