CN109457037A - A kind of genital tract causal agent kit for detecting nucleic acid - Google Patents
A kind of genital tract causal agent kit for detecting nucleic acid Download PDFInfo
- Publication number
- CN109457037A CN109457037A CN201811246232.XA CN201811246232A CN109457037A CN 109457037 A CN109457037 A CN 109457037A CN 201811246232 A CN201811246232 A CN 201811246232A CN 109457037 A CN109457037 A CN 109457037A
- Authority
- CN
- China
- Prior art keywords
- detection
- primer
- probe
- seq
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of genital tract causal agent kit for detecting nucleic acid, including being added after test sample fluorescence quantitative PCR detection is carried out simultaneously and in the first detection components, the second detection components and the third detection components of lyophilised state, first detection components are to detect chlamydia trachomatis and NEISSERIA GONORRHOEAE, second detection components are to detect Ureaplasma urealyticum and mycoplasma hominis, and third detection components are to detect mycoplasma genitalium.The present invention can detect 5 kinds of genital tract chlamydia trachomatis, NEISSERIA GONORRHOEAE, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium nucleic acid simultaneously, the detection of above-mentioned 5 kinds of pathogenic microorganisms is integrated in a consumptive material, lyophilized technique is used simultaneously, reagent stability is improved, single part is disposable, only need to once sample, single stepping, experiment operator is greatly liberated, testing cost is reduced, while reducing patient's inconvenience, mitigates patient economy burden.
Description
Technical field
The invention belongs to molecular diagnostic techniques fields, and in particular to a kind of genital tract causal agent kit for detecting nucleic acid.
Background technique
Infertility, which refers to, does not take contraceptives for 1 year, and sexual life is normal and the gestation that fails.Studies have shown that infertility
Annoying the couple at child-bearing age in the whole world about 16%.Investigation both domestic and external shows that the Tubal factor in biological factor is to cause
The primary factor of Chinese infertility.And the infection of genital tract causal agent is to lead to infertile principal element.Mycoplasma, clothing are former
Body and gonococcus are the main pathogenic fungis of reproductive tract infection, can cause Male urethritis, women vaginitis, cervicitis, pelvic inflammatory disease
And infertility etc..
Sexually transmitted disease caused by chlamydia trachomatis (CT), which is that the whole world is main in recent years, leads to spreading through sex intercourse for infertility
Disease.The main pathological change of CT infection is chronic inflammation, and preinfection may be without obvious clinical symptoms, but with infection
Development, can cause tissue damage, the later period can also form scar, impact to the normal function of organ.
NEISSERIA GONORRHOEAE (NG) is the pathogen of stranguria syndrome, can cause the damage of mucosal epithelium, leads to salpingemphraxis, glues
Even, to influence the normal secretions mechanism of mucosal epithelium, cause infertile.Gonococcus property epididymo-orchitis can lead to duration
Aspermia or oligospermia, azoospermatism.
Infecting in the mycoplasma of genital tract mainly includes Ureaplasma urealyticum (UU), mycoplasma hominis (MH) and genitals branch
Substance (MG) three types.Mycoplasma can be formed on genital tract cells surface to be sticked, and kill drawing to normal cell
The generation for playing endometrium, fallopian tubal chronic inflammation, after Fallopian tube mucosa is damaged, will cause fallopian tube adhesion or obstruction；
In addition, UU, MH and MG also affect to the movement of fallopian tubal cilium, the formation of fertilized eggs is hindered, so as to cause not
The generation of pregnant disease.
The method for being clinically used for reproductive tract infection pathogen detection at present has culture technique, immunological technique and round pcr etc.,
These technologies have played huge effect in clinical diagnosis, but there are still some disadvantages.Culture technique is cumbersome and time-consuming；Exempt from
Epidemic disease technology will have special antiserum.The superiority of round pcr itself is unblamable, but since experimental procedure is more,
And PCR aerosol easily pollutes, and leads to serious false positive results, necessary instrument is expensive, and consumptive material takes opening to set
Meter considers biological safety inadequate.
Summary of the invention
It is an object of the invention to overcome prior art defect, a kind of genital tract causal agent kit for detecting nucleic acid is provided.
Technical scheme is as follows:
A kind of genital tract causal agent kit for detecting nucleic acid, including quantitative fluorescent PCR is carried out simultaneously after test sample is added
The first detection components, the second detection components and the third detection components of lyophilised state are detected and are in, the first detection components are to examine
Chlamydia trachomatis and NEISSERIA GONORRHOEAE are surveyed, the second detection components are to detect Ureaplasma urealyticum and mycoplasma hominis, third detection
Component is to detect mycoplasma genitalium；
Wherein, first to third detection components by trehalose, mannitol, PEG and detection agent composition mixing after be lyophilized
Be made, in first to third detection components detection agent composition by 10 × PCR buffer, dNTPs, detection primer to, it is interior
Join primer pair, detection probe, internal reference probe, MgCl2, BSA, r-Taq enzyme, UNG enzyme and water composition, the amount of trehalose is corresponding
Detection agent composition 1.8N11%, the amount of mannitol is that the amount of 1.5~5%, PEG of corresponding detection agent composition is
The 0.5~2.5% of corresponding detection agent composition, above-mentioned percentage are quality percent by volume, and BSA is in corresponding inspection
The concentration surveyed in agent composition is 0.01~0.03mg/mL, and the molecular weight of above-mentioned PEG is 5500~8000.
In a preferred embodiment of the invention, in first detection components, detection primer is to by CT primer
To and NG primer pair composition, detection probe is made of CT probe and NG probe, and the CT primer pair is by respectively such as SEQ ID NO01
With 02 shown in CT upstream primer and CT downstream primer composition, the NG primer pair is as respectively as shown in SEQ ID NO 04 and 05
NG upstream primer and NG downstream primer composition, the sequence of the CT probe is as shown in SEQ ID NO 03, and the sequence of the NG probe is such as
Shown in SEQ ID NO 06.
In a preferred embodiment of the invention, in second detection components, detection primer is to by MH primer
To and UU primer pair composition, detection probe is made of MH probe and UU probe, and the MH primer pair is by respectively such as SEQ ID NO
MH upstream primer shown in 07 and 08 and MH downstream primer composition, the UU primer pair is as respectively as shown in SEQ ID NO 10 and 11
UU upstream primer and UU downstream primer composition, the sequence of the MH probe is as shown in SEQ ID NO 09, the sequence of the UU probe
As shown in SEQ ID NO 12.
In a preferred embodiment of the invention, in the third detection components, detection primer ties up MG primer pair,
Its detection probe is MG probe, and the MG primer pair is as the MG upstream primer as shown in SEQ ID NO 13 and 14 and the downstream MG respectively
Primer composition, the sequence of the MG probe is as shown in SEQ ID NO 15.
In a preferred embodiment of the invention, the internal control primer is to by respectively such as 16 and 17 institute of SEQ ID NO
Internal reference upstream primer and the internal reference downstream primer composition shown, the internal reference probe is as shown in SEQ ID NO 18.
It is further preferred that described first into third detection components, the amount of trehalose is that corresponding detection agent combines
The 2~10% of object, the amount that the amount of mannitol is 2~5%, PEG of corresponding detection agent composition are corresponding detection agent
The 0.5~2% of composition, above-mentioned percentage are quality percent by volume.
Still more preferably, described first into third detection components, and BSA is in corresponding detection agent composition
Concentration is 0.01~0.02mg/mL.
Still more preferably, the molecular weight of the PEG is 6000~7000.
The beneficial effects of the present invention are: the present invention can detect genital tract chlamydia trachomatis, NEISSERIA GONORRHOEAE, solution urea branch simultaneously
5 kinds of substance, mycoplasma hominis and mycoplasma genitalium nucleic acid, the detection of above-mentioned 5 kinds of pathogenic microorganisms is integrated in a consumptive material,
Lyophilized technique is used simultaneously, improves reagent stability, and single part is disposable, only need to once sample, single stepping, greatly
Experiment operator has been liberated, testing cost is reduced, while having reduced patient's inconvenience, has mitigated patient economy burden.
Detailed description of the invention
Fig. 1 is the fluorescent PCR figure that chlamydia trachomatis is individually detected in the embodiment of the present invention 2.
Fig. 2 is chlamydia trachomatis bacterium solution fluorescent PCR figure in the embodiment of the present invention 2.
Fig. 3 is the fluorescent PCR figure that NEISSERIA GONORRHOEAE is individually detected in the embodiment of the present invention 2.
Fig. 4 is NEISSERIA GONORRHOEAE bacterium solution fluorescent PCR figure in the embodiment of the present invention 2.
Fig. 5 is the fluorescent PCR figure that Ureaplasma urealyticum is individually detected in the embodiment of the present invention 2.
Fig. 6 is Ureaplasma urealyticum bacterium solution fluorescent PCR figure in the embodiment of the present invention 2.
Fig. 7 is the fluorescent PCR figure that mycoplasma hominis is individually detected in the embodiment of the present invention 2.
Fig. 8 is mycoplasma hominis bacterium solution fluorescent PCR figure in the embodiment of the present invention 2.
Fig. 9 is the fluorescent PCR figure that mycoplasma genitalium is individually detected in the embodiment of the present invention 2.
Figure 10 is mycoplasma genitalium bacterium solution fluorescent PCR figure in the embodiment of the present invention 2.
Figure 11 is the testing result figure of sample stoste 1 in the embodiment of the present invention 3.
Figure 12 is the testing result figure of sample stoste 2 in the embodiment of the present invention 3.
Specific embodiment
Technical solution of the present invention is further explained and described below by way of specific embodiment.
Embodiment 1
A kind of genital tract causal agent kit for detecting nucleic acid, including quantitative fluorescent PCR is carried out simultaneously after test sample is added
The first detection components, the second detection components and the third detection components of lyophilised state are detected and are in, the first detection components are to examine
Chlamydia trachomatis and NEISSERIA GONORRHOEAE are surveyed, the second detection components are to detect Ureaplasma urealyticum and mycoplasma hominis, third detection
Component is to detect mycoplasma genitalium；
Wherein, first to third detection components by trehalose, mannitol, PEG and detection agent composition mixing after be lyophilized
Be made, in first to third detection components detection agent composition by 10 × PCR buffer, dNTPs, detection primer to, it is interior
Join primer pair, detection probe, internal reference probe, MgCl2, BSA, r-Taq enzyme, UNG enzyme and water composition, the amount of trehalose is corresponding
Detection agent composition 2~10%, the amount of mannitol is that the amount of 2~5%, PEG of corresponding detection agent composition is institute
The 0.5~2% of corresponding detection agent composition, above-mentioned percentage are quality percent by volume, and BSA is in corresponding detection agent
Concentration in composition is 0.01~0.02mg/mL, and the molecular weight of above-mentioned PEG is 6000~7000.
In first detection components, detection primer is formed to by CT primer pair and NG primer pair, detection probe by
CT probe and NG probe composition, the CT primer pair is as the CT upstream primer as shown in SEQ ID NO 01 and 02 and the downstream CT respectively
Primer composition, the NG primer pair is as the NG upstream primer as shown in SEQ ID NO 04 and 04~6 respectively and NG downstream primer group
At the sequence of the CT probe is as shown in SEQ ID NO 03, and the sequence of the NG probe is as shown in SEQ ID NO 06.
In second detection components, detection primer is formed to by MH primer pair and UU primer pair, detection probe by
MH probe and UU probe composition, the MH primer pair is as the MH upstream primer as shown in SEQ ID NO 07 and 08 and the downstream MH respectively
Primer composition, the UU primer pair is as the UU upstream primer as shown in SEQ ID NO 8~11 and 11 respectively and UU downstream primer group
At the sequence of the MH probe is as shown in SEQ ID NO 09, and the sequence of the UU probe is as shown in SEQ ID NO 12.
In the third detection components, detection primer ties up MG primer pair, and detection probe is MG probe, the MG primer pair
It is made of the MG upstream primer as shown in SEQ ID NO 13 and 14 and MG downstream primer respectively, the sequence of the MG probe such as SEQ
Shown in ID NO 14~6.
The internal control primer is drawn to as the internal reference upstream primer as shown in SEQ ID NO 16 and 17 and internal reference downstream respectively
Object composition, the internal reference probe is as shown in SEQ ID NO 18.
Preferably, the formula before first to the freeze-drying of third detection components is successively as shown in the following table 1 to table 3, and the following table 1 is extremely
The total volume of table 3 is 1000 μ L, i.e., everyone usage amount is 25 μ L:
Table 1
Table 2
| Component | Concentration | Volume (/mL) |
| PCR buffer | 10× | 244~6~260 |
| dNTPs | 8~11mM | 111~114~6 |
| MH/UU/ internal reference upstream primer | 4~60 μM | 8~11 |
| MH/UU/ internal reference downstream primer | 4~60 μM | 8~11 |
| MH/UU/ internal reference probe | 4~60 μM | 4~6 |
| MgCl2 | 24~28mM | 170~178 |
| BSA | 480~550mg/mL | 12~14 |
| PEG-6000 | 480~550mg/mL | 12~14 |
| Trehalose | 480~550mg/mL | 70~90 |
| Mannitol | 480~550mg/mL | 12~14 |
| R-Taq enzyme | 4.5~5U/ μ L | 37~38.5 |
| UNG enzyme | 1.8~2.1U/ μ L | 2~3 |
| Deionized water | Mending to total volume is 1000 |
Table 3
The process of above-mentioned freeze-drying is freeze-dried using the new sesame original position type freeze drier (Scientz-10ND) in Ningbo,
Program is as shown in table 4 below:
Table 4
| Temperature | Time | Whether vacuumize |
| -50℃ | 6 hours | It is no |
| -45℃ | 3 hours | It is |
| -40℃ | 3 hours | It is |
| -35℃ | 2 hours | It is |
| -30℃ | 2 hours | It is |
| -25℃ | 1 hour | It is |
| -20℃ | 1 hour | It is |
| -15℃ | 1 hour | It is |
| -10℃ | 1 hour | It is |
| -5℃ | 1 hour | It is |
| 0℃ | 1 hour | It is |
| 10℃ | 1 hour | It is |
| 20℃ | 1 hour | It is |
The detection process of the genital tract causal agent kit for detecting nucleic acid of the present embodiment is as follows: first to third detection group
Sample to be tested is added in part, is then placed in fluorescence quantitative PCR instrument simultaneously and carries out augmentation detection: initial denaturation according to following amplification program
95℃3min；Expand 40 circulations, 95 DEG C of 15s, 60 DEG C of 30s (acquisition fluorescence)；End-point analysis.
Embodiment 2
It is former that chlamydia trachomatis (CT), genitals branch are detected respectively with 1 genital tract causal agent kit for detecting nucleic acid of embodiment
(pathogen dry powder is all purchased from for body (MG), Ureaplasma urealyticum (UU), NEISSERIA GONORRHOEAE (NG) and mycoplasma hominis (MH) pathogen
ATCC, redissolving respectively is 106The bacterium solution of a/mL), 100~200 μ L bacterium are specially added into third detection components first
Liquid is placed in fluorescence quantitative PCR instrument and carries out augmentation detection: 95 DEG C of 3min of initial denaturation according to following amplification program；Amplification 40 follows
Ring, 95 DEG C of 15s, 60 DEG C of 30s (acquisition fluorescence)
Wherein individually the fluorescent PCR figure of detection chlamydia trachomatis (CT) is as depicted in figs. 1 and 2, individually detects genitals branch
The fluorescent PCR figure of substance (MG) is as shown in Figure 3 and Figure 4, individually detects fluorescent PCR the figure such as Fig. 5 and Fig. 6 of Ureaplasma urealyticum (UU)
Shown, the fluorescent PCR figure for individually detecting NEISSERIA GONORRHOEAE (NG) is as shown in Figure 7 and Figure 8, individually detects mycoplasma hominis (MH) disease
The fluorescent PCR figure of substance is as shown in Figure 9 and Figure 10.
Embodiment 3
The patient from healthcare hospital for women & children, Xiamen City is detected respectively with 1 genital tract causal agent kit for detecting nucleic acid of embodiment
Vagina cervical secretions swab sample 1 and swab sample 2 impregnate swab sample 1, swab sample 2 with 1mL physiological saline respectively,
120s is shaken, sample stoste 1 and sample stoste 2 are obtained.
It is separately added into 200~500 μ L sample stostes 1 into third detection components first, closes the lid, is packed into mating complete
In automatic PCR analysis system, operation 40~60min acquisition testing result is as follows, i.e. NEISSERIA GONORRHOEAE (NG) positive is (such as Figure 11 institute
Show).
It is separately added into 200~500 μ L sample stostes 2 into third detection components first, closes the lid, is packed into mating complete
In automatic PCR analysis system, operation 40~60min acquisition testing result is as follows, i.e. mycoplasma hominis (MH) positive is (such as Figure 12 institute
Show).
Above-mentioned swab sample 1 and 2 is detected with traditional Sanger sequencing simultaneously, about needs to obtain for one week identical
Testing result.
The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e.,
Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.
Sequence table
<110>Merrill Lynch's U.S. nation (Xiamen) Biotechnology Co., Ltd
<120>a kind of genital tract causal agent kit for detecting nucleic acid
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ccgatgaaga ggcgttacga 20
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tggtagagaa ggagcaatcg aaa 23
<210> 3
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttggagtctt cacatgctct cgcacattt 29
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cacggcgatt tcgcagtt 18
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gcacataacg catagcgaaa ttt 23
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
acggcaccat cgtccgtatg gc 22
<210> 7
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ttgcattagg tcagcaacta gctt 24
<210> 8
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gacgttgcaa gaagacgttt agc 23
<210> 9
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
catctcttgc cccttccatt acatgatca 29
<210> 10
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gcgtcatagt tgtccataaa gtcaa 25
<210> 11
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gcaatgcgtt atacagaagc tagaat 26
<210> 12
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
acagcgccac ataaaaaatc ggcaag 26
<210> 13
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cctatgatac caatcctacc ctctca 26
<210> 14
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ttgtaggtgt tagtggtttg gtaagc 26
<210> 15
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ccttccaact ctaccaaccc aacaaggtga 30
<210> 16
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gctctaccgg aaacgtcttt aact 24
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
caccatcccg atttgttcct 20
<210> 18
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
acagggactt gtgatcttgc tcccaacc 28
Claims (8)
1. a kind of genital tract causal agent kit for detecting nucleic acid, it is characterised in that: including being added after test sample while carrying out glimmering
Fluorescent Quantitative PCR detection and the first detection components, the second detection components and third detection components in lyophilised state, the first detection group
Part to detect chlamydia trachomatis and NEISSERIA GONORRHOEAE, the second detection components to detect Ureaplasma urealyticum and mycoplasma hominis,
Third detection components are to detect mycoplasma genitalium；
Wherein, first to third detection components by being lyophilized and be made after the mixing of trehalose, mannitol, PEG and detection agent composition,
First into the detection agent composition of third detection components by 10 × PCR buffer, dNTPs, detection primer to, internal control primer
To, detection probe, internal reference probe, MgCl2, BSA, r-Taq enzyme, UNG enzyme and water composition, the amount of trehalose be corresponding detection
The 1.8~11% of agent composition, the amount that the amount of mannitol is 1.5~5%, PEG of corresponding detection agent composition is right
The 0.5~2.5% of the detection agent composition answered, above-mentioned percentage are quality percent by volume, and BSA is in corresponding detection agent
Concentration in composition is 0.01~0.03mg/mL, and the molecular weight of above-mentioned PEG is 5500~8000.
2. a kind of genital tract causal agent kit for detecting nucleic acid as described in claim 1, it is characterised in that: first detection
In component, detection primer is formed to by CT primer pair and NG primer pair, and detection probe is made of CT probe and NG probe, should
CT primer pair is made of the CT upstream primer as shown in SEQ ID NO 01 and 02 and CT downstream primer respectively, the NG primer pair by
The NG upstream primer as shown in SEQ ID NO 04 and 05 and NG downstream primer composition respectively, the sequence of the CT probe such as SEQ ID
Shown in NO 03, the sequence of the NG probe is as shown in SEQ ID NO 06.
3. a kind of genital tract causal agent kit for detecting nucleic acid as described in claim 1, it is characterised in that: second detection
In component, detection primer is formed to by MH primer pair and UU primer pair, and detection probe is made of MH probe and UU probe, should
MH primer pair is made of the MH upstream primer as shown in SEQ ID NO 07 and 08 and MH downstream primer respectively, the UU primer pair by
The UU upstream primer as shown in SEQ ID NO 10 and 11 and UU downstream primer composition respectively, the sequence of the MH probe such as SEQ ID
Shown in NO 09, the sequence of the UU probe is as shown in SEQ ID NO 12.
4. a kind of genital tract causal agent kit for detecting nucleic acid as described in claim 1, it is characterised in that: the third detection
In component, detection primer ties up MG primer pair, and detection probe is MG probe, and the MG primer pair is by respectively such as SEQ ID NO 13
With 14 shown in MG upstream primer and MG downstream primer composition, the sequence of the MG probe is as shown in SEQ ID NO 15.
5. a kind of genital tract causal agent kit for detecting nucleic acid as described in claim 1, it is characterised in that: the internal control primer
It is formed to by the internal reference upstream primer as shown in SEQ ID NO 16 and 17 and internal reference downstream primer respectively, the internal reference probe is such as
Shown in SEQ ID NO 18.
6. a kind of genital tract causal agent kit for detecting nucleic acid as described in any claim in claim 1 to 5, feature
Be: described first into third detection components, and the amount of trehalose is the 2~10% of corresponding detection agent composition, sweet dew
The amount of alcohol is that the amount of 2~5%, PEG of corresponding detection agent composition is the 0.5~2% of corresponding detection agent composition,
Above-mentioned percentage is quality percent by volume.
7. a kind of genital tract causal agent kit for detecting nucleic acid as claimed in claim 6, it is characterised in that: described first to the
In three detection components, concentration of the BSA in corresponding detection agent composition is 0.01~0.02mg/mL.
8. a kind of genital tract causal agent kit for detecting nucleic acid as claimed in claim 6, it is characterised in that: point of the PEG
Son amount is 6000~7000.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811246232.XA CN109457037A (en) | 2018-10-24 | 2018-10-24 | A kind of genital tract causal agent kit for detecting nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811246232.XA CN109457037A (en) | 2018-10-24 | 2018-10-24 | A kind of genital tract causal agent kit for detecting nucleic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109457037A true CN109457037A (en) | 2019-03-12 |
Family
ID=65608309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811246232.XA Pending CN109457037A (en) | 2018-10-24 | 2018-10-24 | A kind of genital tract causal agent kit for detecting nucleic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109457037A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157820A (en) * | 2019-04-28 | 2019-08-23 | 深圳市国赛生物技术有限公司 | A kind of primer, probe and kit detecting chlamydia trachomatis |
| CN113652493A (en) * | 2021-08-17 | 2021-11-16 | 厦门市妇幼保健院（厦门市计划生育服务中心） | Reproductive tract PCR system capable of reducing PCR competition of three fluorescence channels |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120004113A1 (en) * | 2008-11-25 | 2012-01-05 | Goodgene Inc. | Dna chip, kit for detecting or genotyping bacteria causing sexually transmitted diseases, genotyping antibacterial drug resistance and detecting or genotyping method using the same |
| CN102888464A (en) * | 2012-10-25 | 2013-01-23 | 苏州华益美生物科技有限公司 | Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof |
| CN105392896A (en) * | 2013-03-15 | 2016-03-09 | 达雅高生物科技有限公司 | Rapid and sensitive genotype identification and nucleic acid detection |
-
2018
- 2018-10-24 CN CN201811246232.XA patent/CN109457037A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120004113A1 (en) * | 2008-11-25 | 2012-01-05 | Goodgene Inc. | Dna chip, kit for detecting or genotyping bacteria causing sexually transmitted diseases, genotyping antibacterial drug resistance and detecting or genotyping method using the same |
| CN102888464A (en) * | 2012-10-25 | 2013-01-23 | 苏州华益美生物科技有限公司 | Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof |
| CN105392896A (en) * | 2013-03-15 | 2016-03-09 | 达雅高生物科技有限公司 | Rapid and sensitive genotype identification and nucleic acid detection |
Non-Patent Citations (2)
| Title |
|---|
| GYEONG-IN LEE等: "a comparison of oligonucleotide-based microarray and real-time PCR for the detection of sexually transmitted infections", 《BIOCHIP》 * |
| 王辉等: "多重PCR结合反向线点杂交方法检测七种性病病原体", 《中华皮肤科杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157820A (en) * | 2019-04-28 | 2019-08-23 | 深圳市国赛生物技术有限公司 | A kind of primer, probe and kit detecting chlamydia trachomatis |
| CN113652493A (en) * | 2021-08-17 | 2021-11-16 | 厦门市妇幼保健院（厦门市计划生育服务中心） | Reproductive tract PCR system capable of reducing PCR competition of three fluorescence channels |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sweeney et al. | The human Ureaplasma species as causative agents of chorioamnionitis | |
| Eckert et al. | The antimicrobial treatment of subacute endometritis: a proof of concept study | |
| Mitchell et al. | Bacterial vaginosis and the cervicovaginal immune response | |
| Hanna et al. | The relation between vaginal pH and the microbiological status in vaginitis | |
| Polisseni et al. | Detection of chronic endometritis by diagnostic hysteroscopy in asymptomatic infertile patients | |
| Chappell et al. | The effects of reproductive hormones on the physical properties of cervicovaginal fluid | |
| CN100439516C (en) | Pig breeding and respiration syndrome virus ultrastrong variation strain RT-PCR reagent case | |
| CN102719529B (en) | Trichomonas vaginalis and candida albicans two-channel fluorescence PCR detection method and kit thereof | |
| Roomruangwong et al. | The menstrual cycle may not be limited to the endometrium but also may impact gut permeability | |
| CN109457037A (en) | A kind of genital tract causal agent kit for detecting nucleic acid | |
| Chaplin et al. | The human microbiome | |
| CN110343780A (en) | Gardner bacillus, the primer of Candida albicans and trichomonas vaginalis Multiple detection, probe groups, kit and detection method | |
| CN102212623A (en) | Two-color fluorescence quantitative polymerase chain reaction (PCR) combined detection method of swine fever virus and blue ear disease virus and kit thereof | |
| Gao et al. | An updated review of feline coronavirus: Mind the two biotypes | |
| CN108504782A (en) | Primer combination for 4 boar infectious disease viruses of synchronous detection and detection kit | |
| CN101608210A (en) | Quantitative detection kit for helicobacter pylori nucleic acid | |
| CN110343775A (en) | The primer of double check Gardner bacillus and trichomonas vaginalis, probe groups, kit and detection method | |
| CN104513854B (en) | A kind of pathogen nucleic acid and drug resistance gene detection kit and its application | |
| Holdcroft et al. | The Vaginal Microbiome in Health and Disease—What Role Do Common Intimate Hygiene Practices Play? Microorganisms 2023, 11, 298 | |
| El-Mahgoub | Antispermatozoal antibodies in infertile women with cervicovaginal schistosomiasis | |
| Tosun et al. | Frequency of bacterial vaginosis among women attending for intrauterine device insertion at an inner-city family planning clinic | |
| Stamey et al. | Studies of introital colonization in women with recurrent urinary infections. III. Vaginal glycogen concentrations | |
| CN108707587A (en) | One plant of Europe class porcine reproductive and respiratory syndrome virus strain and its application | |
| Gumenyuk et al. | GUT MICROBIOTA ALTERATIONS AND THEIR ASSOCIATION WITH IL6, IL8 AND TNFα LEVELS IN PATIENTS WITH EXTERNAL GENITAL ENDOMETRIOSIS | |
| Hillier et al. | O05. 1 Lower genital tract predictors of acute endometritis among women with signs and symptoms of pelvic inflammatory disease (PID) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190312 |