CN110257534A - Ureaplasma urealyticum kit for detecting nucleic acid - Google Patents
Ureaplasma urealyticum kit for detecting nucleic acid Download PDFInfo
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- 241000202921 Ureaplasma urealyticum Species 0.000 title claims abstract description 50
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 40
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 40
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 40
- 238000001514 detection method Methods 0.000 claims abstract description 60
- 239000000523 sample Substances 0.000 claims abstract description 33
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 14
- 238000010791 quenching Methods 0.000 claims abstract description 9
- 230000000171 quenching effect Effects 0.000 claims abstract description 9
- 125000006853 reporter group Chemical group 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 7
- 238000007400 DNA extraction Methods 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 4
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- 229940093429 polyethylene glycol 6000 Drugs 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical group [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 8
- -1 upstream primer Chemical class 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 15
- 238000000034 method Methods 0.000 description 9
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- 241000701806 Human papillomavirus Species 0.000 description 4
- 241000204051 Mycoplasma genitalium Species 0.000 description 4
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 4
- 241000588653 Neisseria Species 0.000 description 4
- 229940038705 chlamydia trachomatis Drugs 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
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- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 206010056254 Intrauterine infection Diseases 0.000 description 1
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- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
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- 210000002826 placenta Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- Analytical Chemistry (AREA)
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- Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
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Abstract
The present invention discloses a kind of ureaplasma urealyticum kit for detecting nucleic acid, including upstream primer, downstream primer and probe, and the sequence of the upstream primer is as shown in SEQ ID NO:1, and the sequence of the downstream primer is as shown in SEQ ID NO:2；The probe is x-CCATCAGGTACTGCTATTCG-MGB；Alternatively, the probe is x-CCATCAGGTACTGCTATTCG-y+MGB, the x is fluorescent reporter group, and the y is quenching group.Technical solution of the present invention has the advantages that detection sensitivity is good, detection accuracy is high and detection operation is convenient.
Description
Technical Field
The invention relates to the field of detection, and particularly relates to a ureaplasma urealyticum nucleic acid detection kit.
Background
Ureaplasma Urealyticum (UU) is one of the common parasitic bacteria in the human urogenital tract and can cause diseases under specific environments. UU has simple structure, no cell wall, high polymorphism, high nutritive requirement and slow growth. UU has two peak periods in the field planting quantity of human body, namely, the newborn is infected by the maternal birth canal during delivery, and then the UU is rapidly reduced and gradually increased from the beginning of sexual life. UU is transmitted mainly through sexual life, or vertically through placenta or upward spread from infection of the lower genital tract of pregnant women, causing intrauterine infection. Its infection can lead to infertility, abortion in pregnant women, premature birth, retarded fetal development, and other adverse consequences. At present, the etiology examination method mainly comprises separation culture and molecular biology detection, different methods have the characteristics, and the method for rapidly, simply, conveniently and accurately detecting UU is very necessary for clinical diagnosis and treatment.
Disclosure of Invention
The invention mainly aims to provide a ureaplasma urealyticum nucleic acid detection kit, and aims to improve the detection precision of ureaplasma urealyticum nucleic acid detection.
In order to realize the purpose, the ureaplasma urealyticum nucleic acid detection kit provided by the invention comprises an upstream primer, a downstream primer and a probe, wherein the sequence of the upstream primer is shown as SEQ ID NO. 1, and the sequence of the downstream primer is shown as SEQ ID NO. 2;
the probe is x-CCATCAGGTACTGCTATTCG-MGB; or,
the probe is x-CCATCAGGTACTGCTATTCG-y + MGB, wherein x is a fluorescent reporter group, and y is a quenching group.
Optionally, in this embodiment, x is any one of a FAM group, a VIC group, and a TEXAS RED group.
Optionally, the ureaplasma urealyticum nucleic acid detection kit further comprises Taq enzyme, PCR buffer solution, dNTPS and MgCl2。
Optionally, the ureaplasma urealyticum nucleic acid detection kit further comprises a DNA extraction solution, and the DNA extraction solution comprises: polyethylene glycol 6000, sodium chloride, sodium dodecyl sulfate Tween-20, ethanol and a TE buffer solution serving as a solvent.
The technical scheme of the invention adopts the upstream primer with the sequence shown as SEQ ID NO. 1, the downstream primer with the sequence shown as SEQ ID NO. 2 and the probe to detect ureaplasma urealyticum nucleic acid, and the upstream primer and the downstream primer are positioned in a gene conserved region, so that all serotypes can be amplified, and the invention has the advantage of wide application range. The probe is an MGB probe, a quenching group of the MGB probe adopts a non-fluorescence quenching group, and the non-fluorescence quenching group does not generate fluorescence, so that the intensity of a background signal can be greatly reduced, and the sensitivity of fluorescence change during detection can be improved, thereby improving the sensitivity of detection; on the other hand, the MGB group can improve the Tm value of the probe by about 10 ℃, which is beneficial to reducing nonspecific combination and improving the detection precision. As long as there is a base mismatch between the probe and the template, the probe can detect the base mismatch, and the probe can not hybridize with the target fragment, so that a fluorescent signal is not generated, the detection specificity is greatly improved, and the detection precision is favorably improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a graph showing fluorescence intensity variation curves of Mycoplasma hominis, human papilloma virus, Chlamydia trachomatis, gonococcus, cytomegalovirus, Mycoplasma pneumoniae, Mycoplasma genitalium, herpes simplex virus type II, Escherichia coli, ureaplasma urealyticum standard strains, ureaplasma urealyticum clinical strains and positive quality control substances tested by using an embodiment of the ureaplasma urealyticum nucleic acid detection kit of the present invention, wherein S-shaped curves 1 to 3 in FIG. 1 are fluorescence intensity variation curves of the ureaplasma urealyticum standard strains, the ureaplasma urealyticum clinical strains and the positive quality control substances in sequence;
FIG. 2 shows that the concentrations of the nucleic acid detecting kit for ureaplasma urealyticum according to an embodiment of the present invention are 106copies/μL、105copies/μL、104copies/μL、103copies/μL、102Fluorescence intensity curves of the templates of copies/μ L and 20copies/μ L, wherein S-shaped curves 4 to 9 in FIG. 2 are 10 in sequence respectively6copies/μL、105copies/μL、104copies/μL、103copies/μL、102Fluorescence intensity profiles for copies/. mu.L, 20 copies/. mu.L concentration templates.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that, if directional indications (such as up, down, left, right, front, and back … …) are involved in the embodiment of the present invention, the directional indications are only used to explain the relative positional relationship between the components, the movement situation, and the like in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indications are changed accordingly.
In addition, if there is a description of "first", "second", etc. in an embodiment of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
The invention provides a ureaplasma urealyticum nucleic acid detection kit.
In the embodiment of the invention, in order to realize the purpose, the ureaplasma urealyticum nucleic acid detection kit provided by the invention comprises an upstream primer, a downstream primer and a probe, wherein the sequence of the upstream primer is shown as SEQ ID NO. 1, and the sequence of the downstream primer is shown as SEQ ID NO. 2; the probe is x-CCATCAGGTACTGCTATTCG-y + MGB, wherein x is a fluorescent reporter group, and y is a quenching group. When the ureaplasma urealyticum nucleic acid detection kit is used for testing, firstly, the DNA in a to-be-tested object is extracted, then, the DNA obtained by testing is tested by adopting the upstream primer, the downstream primer and the probe, and then, fluorescence is detected to obtain a detection result. The probe described in this embodiment is not limited to the above technical solution, and in other embodiments, the probe may be x-CCATCAGGTACTGCTATTCG-MGB, and x is a fluorescent reporter group.
The technical scheme of the invention adopts the upstream primer with the sequence shown as SEQ ID NO. 1, the downstream primer with the sequence shown as SEQ ID NO. 2 and the probe to detect ureaplasma urealyticum nucleic acid, and the upstream primer and the downstream primer are positioned in a gene conserved region, so that all serotypes can be amplified, and the invention has the advantage of wide application range. The probe is an MGB probe, a quenching group of the MGB probe adopts a Non-Fluorescent quenching group (Non-Fluorescent Quencher), and does not generate fluorescence, so that the intensity of a background signal can be greatly reduced, the sensitivity of fluorescence change during detection can be improved, and the detection sensitivity can be improved; on the other hand, the MGB group can improve the Tm value of the probe by about 10 ℃, which is beneficial to reducing nonspecific combination and improving the detection precision. As long as there is a base mismatch between the probe and the template, the probe will detect the base mismatch, and the probe will not hybridize with the target fragment, thereby generating no fluorescent signal and greatly improving the specificity of detection.
Further, in this embodiment, x is any one of a FAM group, a VIC group, and a TEXAS RED group, and has a characteristic of good fluorescence effect.
Further, in this embodiment, the ureaplasma urealyticum nucleic acid detection kit further includes Taq enzyme, PCR buffer, dNTPS, and MgCl2. Taq enzyme is DNA polymerase with good heat resistance, can endure the high temperature of more than 90 ℃ without inactivation, can be continuously used in the whole amplification process, and leads the amplification process to be very simple and rapid. The ureaplasma urealyticum nucleic acid detection kit also comprises UNG enzyme.
Further, in this embodiment, the ureaplasma urealyticum nucleic acid detection kit further includes a DNA extraction solution, and the DNA extraction solution includes: polyethylene glycol 6000, sodium chloride, sodium dodecyl sulfate Tween-20, ethanol and a TE buffer solution serving as a solvent.
The use method of the ureaplasma urealyticum nucleic acid detection kit comprises the following steps:
putting 200 mu L of secretion into a 1.5mL centrifuge tube, centrifuging for 5 minutes at the rotating speed of 12000rpm, and removing liquid to obtain a substrate;
adding a DNA extracting solution into the substrate, heating at 100 ℃ for 10 minutes, centrifuging again, and obtaining a supernatant to obtain the sample nucleic acid; the DNA extracting solution specifically comprises: 50mM/L polyethylene glycol 6000, 100mM/L sodium chloride, 0.2 wt.% sodium dodecyl sulfate, 0.5 wt.% Tween-20, 0.1 wt.% ethanol and a solvent TE buffer;
adding 40 mu L of fluorescent PCR system into the sample nucleic acid, heating for 3min at 95 ℃, 15 seconds at 95 ℃ and 35 seconds at 52 ℃ to form a heating cycle, and heating for 40 heating cycles; the 40 mu L fluorescent PCR system comprises 1.5U-2U Taq enzyme, 0.5U UNG enzyme, PCR buffer solution, 0.2mM dNTPS and 2mM MgCl20.2 μ M upstream primer, 0.2 μ M downstream primer and 0.1 μ M probe; one heating cycle for each PCR reactionA period; the molar ratio of each component in the dNTPS is dATP: dCTP to dGTP: dUTP is 1:1:1: 1.
The change in fluorescence during the heating cycle is monitored and if the fluorescence signal increases, a ureaplasma urealyticum viral infection is present.
The human mycoplasma, human papilloma virus, chlamydia trachomatis, gonococcus, cytomegalovirus, mycoplasma pneumoniae, mycoplasma genitalium, herpes simplex virus II and Escherichia coli are common pathogens or colonizing bacteria of human urogenital tract, and are pathogens or colonizing bacteria which are often accompanied with ureaplasma urealyticum nucleic acid when a kit is used for detecting secretion. In order to detect the specificity of the ureaplasma urealyticum nucleic acid detection kit, the use method of the ureaplasma urealyticum nucleic acid detection kit is adopted to detect targets comprising mycoplasma hominis, human papilloma virus, chlamydia trachomatis, gonococcus, cytomegalovirus, mycoplasma pneumoniae, mycoplasma genitalium, herpes simplex virus II, escherichia coli and ureaplasma urealyticum clinical strains, a fluorescence PCR (polymerase chain reaction) instrument is used for monitoring and analyzing fluorescence to judge results, and the change curve of the fluorescence intensity obtained by detection is shown in figure 1.
In FIG. 1, it is shown that after 40 PCR reaction cycles, the fluorescence intensity of the ureaplasma urealyticum clinical strain is greater than that of the positive quality control substance and less than that of the ureaplasma urealyticum standard strain, and the ureaplasma urealyticum nucleic acid detection kit is effective for the detection result of the ureaplasma urealyticum clinical strain. As can be seen from fig. 1, when the ureaplasma urealyticum nucleic acid detection kit of the present embodiment is used for detection, no fluorescence increase occurs in mycoplasma hominis, human papilloma virus, chlamydia trachomatis, gonococcus, cytomegalovirus, mycoplasma pneumoniae, mycoplasma genitalium, herpes simplex virus type ii, and escherichia coli, but a significant fluorescence increase occurs in a ureaplasma urealyticum clinical strain, so that the ureaplasma urealyticum nucleic acid detection kit has good specificity.
In order to detect the specificity of the ureaplasma urealyticum nucleic acid detection kit, the using method of the ureaplasma urealyticum nucleic acid detection kit is adopted, and the concentration is determinedAre respectively 106copies/μL、105copies/μL、104copies/μL、103copies/μL、102The primers/. mu.L and 20 primers/. mu.L of the plasmid ligated with the target gene were detected, the results were analyzed by fluorescence monitoring and analysis using a fluorescence PCR instrument, and the change curve of the fluorescence intensity obtained by detection was shown in FIG. 2, with the lowest detection amount of the fluorescence analysis method being the lower limit of detection.
As can be seen from FIG. 2, the concentration was 106The curve obtained by detection of the template of copies/muL begins to generate fluorescence increase in the 18 th PCR reaction period and generates obvious fluorescence increase in the 20 th PCR reaction period, so that the ureaplasma urealyticum nucleic acid detection kit can judge the detection result according to the fluorescence increase in the 20 th PCR reaction period, the lower limit of the ureaplasma urealyticum nucleic acid detection kit for detecting ureaplasma urealyticum is 20copies, and the ureaplasma urealyticum nucleic acid detection kit has good sensitivity and can effectively improve the detection accuracy; at a concentration of 106copies/μL、105copies/μL、104copies/μL、103copies/μL、102All templates of copies/mu L and 20 copies/mu L have obvious fluorescence increase in 40 PCR reaction cycles, so that the ureaplasma urealyticum nucleic acid detection kit has good sensitivity; the concentration of the detected template is as low as 20 copies/. mu.L, and the detection can be carried out in the 40 th PCR reaction period, and based on good sensitivity, the ureaplasma urealyticum nucleic acid detection kit has the advantage of less detection period number, and can effectively improve the detection efficiency.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen City Spanish Biotechnology Limited
<120> a nucleic acid detection kit for ureaplasma urealyticum
<130> QPA1912443CN
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
cgttgcatta gatggtggta 20
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
aggtgaaaca gttgtggct 19
Claims (4)
1. A ureaplasma urealyticum nucleic acid detection kit is characterized by comprising an upstream primer, a downstream primer and a probe, wherein the sequence of the upstream primer is shown as SEQ ID NO. 1, and the sequence of the downstream primer is shown as SEQ ID NO. 2;
the probe is x-CCATCAGGTACTGCTATTCG-MGB, or,
the probe is x-CCATCAGGTACTGCTATTCG-y + MGB, wherein x is a fluorescent reporter group, and y is a quenching group.
2. The ureaplasma urealyticum nucleic acid detection kit of claim 1, wherein x is any one of FAM group, VIC group, TEXAS RED group.
3. The ureaplasma urealyticum nucleic acid detection kit of claim 1, further comprising Taq enzyme, PCR buffer, dNTPS and MgCl2。
4. The ureaplasma urealyticum nucleic acid detection kit according to any one of claims 1 to 3, further comprising a DNA extraction solution comprising: polyethylene glycol 6000, sodium chloride, sodium dodecyl sulfate Tween-20, ethanol and a TE buffer solution serving as a solvent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703176A (en) * | 2022-03-30 | 2022-07-05 | 东南大学 | DNA probe for detecting ureaplasma urealyticum nucleic acid and application thereof |
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|---|---|---|---|---|
| US6531583B1 (en) * | 1998-01-30 | 2003-03-11 | Uab Research Foundation, The | Nucleic acid probes and method for detecting Ureaplasma urealyticum |
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| CN102409098A (en) * | 2011-11-25 | 2012-04-11 | 泰普生物科学（中国）有限公司 | Ureaplasma urealyticum PCR (Polymerase Chain Reaction) detection kit and detection method thereof |
| CN103074429A (en) * | 2013-01-10 | 2013-05-01 | 湖南圣湘生物科技有限公司 | UU (ureaplasma urealyticum) detection kit |
| CN103361405A (en) * | 2012-04-01 | 2013-10-23 | 上海市肺科医院 | PCR (Polymerase Chain Reaction) kit for detecting ureaplasma urealyticum biogroup |
| CN103882133A (en) * | 2014-03-31 | 2014-06-25 | 深圳意达凯生物科技有限公司 | Primer pair for detecting ureaplasma urealyticum, kit and application thereof |
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