CN102409098A - Ureaplasma urealyticum PCR (Polymerase Chain Reaction) detection kit and detection method thereof - Google Patents
Ureaplasma urealyticum PCR (Polymerase Chain Reaction) detection kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to the field of microorganism detection, in particular to an ureaplasma urealyticum PCR (Polymerase Chain Reaction) detection kit and a detection method thereof, aims to provide the ureaplasma urealyticum PCR detection kit and the detection method thereof for detecting a specific nucleic acid sequence of ureaplasma urealyticum in a clinical sample so as to fulfill the aim of quickly judging the existence of the ureaplasma urealyticum. In order to fulfill the aim, the technical scheme is to provide the ureaplasma urealyticum PCR detection kit, which includes DNA (Deoxyribonucleic Acid) extract, PCR reaction solution, dNTPs, tap enzyme, UNG enzyme, positive control, negative control and a quantitative reference product, wherein the PCR reaction solution includes a primer A and a fluorescent probe A; and the primer is divided into an upstream primer A and a downstream primer A. The kit has the advantages of high flexibility and specificity, stability, timeliness, convenience in operation and the like.
Description
Technical field
The present invention relates to the microorganism detection field, be specifically related to a kind of ureaplasma urealyticum bacterial strain PCR detection kit and detection method thereof.
Background technology
Ureaplasma urealyticum (Ureaplasmaurealyticum is called for short UU) is one of common bacterial parasite of human urogenital tract, under specific environment, can cause a disease.Field planting at human body can have the secondary ascendant trend, infects the newborn infant by the parent birth canal when promptly giving a birth, and reduces rapidly later on; Begin and cumulative many from sexual life.In recent years, urogenital infections comes into one's own day by day due to the ureaplasma urealyticum, is one of pathogenic agent that causes nongonococcal urethritis.Classified as the pathogenic agent of sexually transmitted disease (STD) at present.
Ureaplasma urealyticum is one of common disease substance of urogenital infections, is generally surface infection, scarcely invades blood.Mechanism of causing a disease is still not fully aware of, and thinking at present maybe be relevant with aggressive enzyme and toxic product.Ureaplasma urealyticum can cause urinary system infection and Infertility under certain condition.The ureaplasma urealyticum multiple parasitism is at male urethra, prepuce of penis and vagina.If up infection, can cause male prostatitis or epididymitis, women vaginitis, cervicitis, and can infect fetus and cause miscarriage, premature labor and under-weight fetus, also can cause the infection of newborn respiration road and central nervous system.
Several kinds of detection methods commonly used:
MDI analytical method: as a kind of method of biopsy; MDI analyzes the means extremely dispute always in the field of business of mycoplasma; There is following deficiency in this technology: the mycoplasma that in genitourinary system, can detect is up to 7 kinds more than, the type of mycoplasma differentiated have difficulties; L-type ne ar and mycoplasma all lack cell walls, and both are difficult to differentiate under mirror; The domestic experimental program that does not still have standard, the stdn of detected result also is left to be desired, but this technology possesses economy, intuitively and characteristic feature quickly.Our experience confirms that with research MDI analyzes and can reduce the inspection cost.Shorten the testing time, and can the direct viewing pathogenic agent, to detecting UU certain application value is arranged, but only be suitable for primary dcreening operation.
Culture method: being main path and the means of finding contagium at present clinically, is the important evidence of ureaplasma urealyticum diagnosis and regimen, is " gold standard " with culture method still at present.The UU solid culture is the gold standard of the detection UU that generally acknowledges.UU is cultivated " national Clinical Laboratory working specification " (the 3rd edition) and the susceptibility method makes explicit provisions.Because solid culture is had relatively high expectations to experimental situation and operator, experimental period is longer, and the susceptibility detection method---agar dilution is more loaded down with trivial details, has limited its clinical application to a certain extent simultaneously.
Immunofluorescence detects: this is the antigenic method of using the earliest of detection, normally on the bacterium colony of solid culture, add special fluorescently-labeled one anti-or two anti-, observations under fluorescent microscope.But the bacterium colony of the fluorescent dye that is not easily distinguishable and undyed bacterium colony; Need special fluorescent microscope, be difficult for popularizing and use; The nonspecific reaction of immunofluorescence antibody can influence shortcomings such as experimental result and experimental result shelf time weak point.
Immunoenzyme detects: this method is similar to the bacterium colony immunofluorescent test, just replaces FLA with enzyme labelled antibody, and operation steps is basic identical, observations under the simple microscope.But the antiserum(antisera) that it needs is more, can wash out bacterium colony in the dyeing course, and antigen antibody reaction influence factor complicacy has all restricted its application.
Immunity combine to detect: this method is development and coming on the basis of immunoenzyme; Its crucial step is to adopt nitrocellulose filter as carrier; Utilize this film to protein adsorptive power preferably, and combine, accomplish mensuration the mycoplasma surface antigen with the immunoenzyme method.But immunity combines the susceptibility of detection not high, and the mycoplasma that is fixed on the NC film reduces special dyeing sometimes and strengthened the cross reaction between each mycoplasma.
Summary of the invention
The present invention seeks to adopt polymerase chain reaction and molecular beacon fluorescent probe technique that high conservative specific nucleic acid sequence in the ureaplasma urealyticum gene is carried out augmentation detection, thereby judge that ureaplasma urealyticum exists, to the auxiliary diagnosis of ureaplasma urealyticum infection.
Technical scheme of the present invention comprises the PCR reaction solution for a kind of ureaplasma urealyticum PCR detection kit is provided, and it is characterized in that, said PCR reaction solution comprises primer and fluorescent probe, and said primer is divided into upstream primer A and downstream primer A:
The nucleotides sequence of said upstream primer A is classified as: 5 '-GCTAAATCAACAGACGGT-3 ';
The nucleotides sequence of said downstream primer A is classified as: 5 '-GCTGCACTTACATCAGCTG-3 ';
The nucleotides sequence of said fluorescent probe A is classified as:
5’-TGCGGTTTACGAAATTGAAAACT-3’。
Preferably, said test kit also comprises DNA extraction liquid, dNTPs, t ap enzyme, UNG enzyme, archaeal dna polymerase, positive control, negative control, quantitative reference article.
Preferably, said dNTPs comprises dATP, dCTP, dGTP, dCTP.
Preferably; Having introduced an artificial constructed internal reference system is used to avoid the false-negative internal reference of detected result system to comprise confidential reference items probe, primer and confidential reference items DNA; Said internal reference system is an Arabidopis thaliana internal reference system; Said internal reference system comprises upstream primer B:SEQ ID NO.5, downstream primer B:SEQ ID NO.6, fluorescent probe: SEQ ID NO.7, specific as follows:
Upstream primer B:5 '-TCATCGTCGCTGGAGCTGGTT-3 '
Downstream primer B:5 '-CGGCGGTTTGTCAAGCTGAT-3 '
Fluorescent probe B:
5′-HEX-CTTCTTATAGTCACTGCACTAAACTGGAT-TAMRA-3′。
Preferably, 5 ' the end flag F AM fluorophor of said fluorescent probe A, 3 ' end mark BHQ quenching group.
Another kind of technical scheme of the present invention is for providing a kind of ureaplasma urealyticum PCR detection method; Wherein the target sequence of pcr amplification is SEQ ID NO.4; The pcr amplification the primer is: the upstream primer sequence is like SEQ ID NO.1; The downstream primer sequence is like SEQ ID NO.2, and the fluorescent probe sequence is like SEQ ID NO.3.
As the further setting of aforesaid method, 5 ' the end flag F AM fluorophor of said fluorescent probe A, 3 ' end mark TAMRA quenching group.
Beneficial effect of the present invention detects for utilizing in the ureaplasma urealyticum gene high conservative specific nucleic acid sequence to carry out nucleic acid amplification, thereby judges the existence of ureaplasma urealyticum.It is effective that this test kit has DNA extraction, weak point simple to operate, consuming time (2～3 hours), and the result is objective reliable, the automatic Collection and analysis data of instrument.Sensitivity and specificity are high.Minimum detectability is 5.0 * 10
2Copies/mL.Used Arabidopis thaliana internal reference system it does not all have homologous gene in ureaplasma urealyticum genome and human genome, and whether to detect in each PCR reaction have PCR inhibition exist, thereby guarantee PCR result's credibility if therefore can be used as internal reference.
Description of drawings
Fig. 1: sensitivity reference article detect figure as a result;
Fig. 2: typical curve reference article detect figure as a result;
Fig. 3: typical curve;
Fig. 4: the UU specificity is figure as a result.
Embodiment
The present invention adopts polymerase chain reaction (PCR) and molecular beacon fluorescent probe (MoleculA.r beacon) technology that high conservative specific nucleic acid sequence in ureaplasma urealyticum (Ureaplasmaurealyticum the is called for short UU) gene is carried out augmentation detection, thereby judges the existence of ureaplasma urealyticum.Thereby patient's medication that direct clinical doctor infects ureaplasma urealyticum helps prognosis to judge.
The conservative specific nucleic acid sequence of above-mentioned height is shown in SEQ ID NO.4.
The internal reference principle: test kit is provided with internal reference gene, i.e. arabidopsis gene.It does not all have homologous gene in ureaplasma urealyticum genome and human genome, whether to detect in each PCR reaction have PCR inhibition exist, thereby guarantee PCR result's credibility if therefore can be used as internal reference.When the internal reference result was sun, expression PCR reaction system and operation were normal; Therefore when the goal gene result was the moon, the internal reference result just seemed very important for sun; But when the goal gene result was sun, the amplification curve of internal reference will be postponed than the goal gene amplification curve, or the internal reference result is normal for Yintu(K19); When yet goal gene and internal reference gene result were cloudy, it is invalid that this experiment is regarded as, and needs to repeat again.
The positive control principle: each test all need be done positive control simultaneously.The positive control result is a sun, shows that the detection system to target gene is normal; And when the result is the moon, show that this time experiment is invalid, need repetition.
The negative control principle: exist for proof has not pollute, each test also need be done negative control simultaneously.The negative control result is cloudy, shows that this test is pollution-free; Like the result is sun, shows that then this time experiment is invalid, needs repetition.
Primer and probe: the applied primer of the present invention, probe all entrust Sigma and Biosearch company to synthesize, and through Sigma quality inspection qualified (containing mass spectrum identifies);
Wherein the optimum primer of UU gene conserved sequence, probe sequence make up as follows:
The nucleotides sequence of said upstream primer A is classified SEQ ID NO.1 as:
5’-GCTAAATCAACAGACGGT-3’；
The nucleotides sequence of said downstream primer A is classified SEQ ID NO.2 as:
5’-GCTGCACTTACATCAGCTG-3’；
The nucleotides sequence of said fluorescent probe A is classified SEQ ID NO.3 as:
5’-TGCGGTTTACGAAATTGAAAACT-3’。
The Arabidopis thaliana internal reference system (Suc) of this test kit, said internal reference system comprises upstream primer B:SEQ ID NO.5, downstream primer B:SEQ ID NO.6, fluorescent probe: SEQ ID NO.7, specific as follows:
Upstream primer B:5 '-TCATCGTCGCTGGAGCTGGTT-3 '
Downstream primer B:5 '-CGGCGGTTTGTCAAGCTGAT-3 '
Fluorescent probe B:
5′-HEX-CTTCTTATAGTCACTGCACTAAACTGGAT-TAMRA-3′。
Embodiment 1
Table 1: test kit is formed
UU-PCR reaction solution prescription is filled a prescription like table 2:
Table 2
Basis reagent preparation:
1.1 Tris: analytical pure, the supplier's of qualified qualification product is arranged, content 99.7%, infrared qualified, pH (5% water) 10.3-10.9, moisture content 0.3%, fusing point 167-171 ℃, absorption system is qualified, and the high-content of impurity is qualified.
1.2 NaOH: analytical pure, Beijing chemical reagent factory's product or the supplier's of qualified qualification product is arranged.
1.3 TritonX100: brown oily liquids can be dissolved in cold water, and the intensive lathering property is arranged.
1.4 EDTA: analytical pure, the supplier's of qualified qualification product is arranged, water-soluble for the white crystals sprills, it is acid that solution is, and is insoluble in alcohol, and content is no less than 99.5%, and reactant aqueous solution is qualified, complexing power stand the test, the high-content of impurity is qualified.
1.5 purified water: use Thymopetidum Injection thing science ltd pure water, handle resistivity 16～18M Ω then through the Milli-Q of Millipore company Biocel type water purification machine.
The preparation of 10mmol/L Tris-HCl solution: between the preparation of production area reagent, take by weighing the Tris of 0.12114g, adding has contained in the 100mL beaker of 60mL zero(ppm) water; Jolting makes it abundant dissolving; Move in the volumetric flask of 100mL, with 10mL distilled water wash beaker 3 times and move in the volumetric flask, use HCl adjust pH is 8.3; At last with the zero(ppm) water constant volume to 100mL, the upset volumetric flask make it abundant mixing.Move into reagent bottle and mark title, concentration, setup time.
The preparation of dNTPs: with dATP, dUTP, dGTP, the dCTP of the 100mM that buys; According to 1: 1.5: 1: 1 (volume ratio) mixed; Each constituent concentration of mixed solution is respectively: 10mM, 15mM, 10mM, 10mM get the 0.5mL centrifuge tube, are distributed into the mother liquor of every pipe 100 μ L dN (U) TP.
The dilution of primer and probe
With the centrifugal 1min of synthetic primer 13000rpm, add an amount of pure water (calculating) primer is dissolved into 100 μ M according to resultant quantity, get the 0.5mL centrifuge tube, be distributed into the primer mother liquor of every pipe 100 μ L.Need to add an amount of pure water before producing and be mixed with 10 μ M use liquid.
With the centrifugal 1min of synthetic probe 13000rpm, add an amount of pure water (calculating) probe is dissolved into 100 μ M according to resultant quantity, get the 0.5mL centrifuge tube, be distributed into the probe mother liquor of every pipe 100 μ L.Need to add an amount of pure water before producing and be mixed with 10 μ M use liquid.
UU DNA extraction liquid: use pure water preparation final concentration to be 10mM Tris-HCl, 25mM NaOH, 1% Triton-100 (v/v), 1% (v/v) NP-40,0.1mM EDTA, 5% (w/v) chelex-100 solution according to turnout; Be sub-packed in the frozen pipe of 2mL with the 1mL/ pipe; Labelled, treat to send quality inspection portion to be carried out to the article quality inspection after the packing of product.
Internal reference
Consist of 10
3Copies/mL contains the internal reference gene plasmid; Receive the high density plasmid that contains the internal reference gene through the spectrophotometer accurate quantification, make 10 times of gradient dilutions to 103copies/mL with 1 * TE, as internal reference, 1mL/ manages packing.
Positive quality control product
Consist of 10
6Copies/mL-10
7Copies/mL contains purpose fragment plasmid; Receive through the ultraviolet spectrophotometer accurate quantification contain the segmental high density plasmid of purpose, with the 1 * TE for preparing in advance do 10 times of gradient dilutions to 106copies/mL-107copies/mL as positive control, 1mL/ manages packing.TE compound method: get 1mol/L Tris-HCl PH 8.010ml, add 0.5mol/LEDTA 2ml, add water to 1000ml.
Weak positive quality control product
Consist of 10
4Copies/mL-10
3Copies/mL contains purpose fragment plasmid; Receive through the ultraviolet spectrophotometer accurate quantification contain the segmental high density plasmid of purpose, make 10 times of gradient dilutions to 10 with the 1 * TE for preparing in advance
4Copies/mL-10
3Copies/mL is as positive control, and 1mL/ manages packing.
Negative quality control product: 1 * TE, 200 μ L/ pipe carries out packing.
The use of embodiment 2 test kits of the present invention
One, reagent is prepared (reagent area in preparation)
1. take out DNA extraction liquid, subsequent use.
2. after confirming that the reaction tubes that need carry out is counted n (number of samples+feminine gender+strong positive quality control product+weak positive quality control product+4 quantitative reference article); Take out the UU-PCR reaction solution, with n * 44 μ lUU-PCR reaction solutions, n * 1 μ l archaeal dna polymerase system adds in the centrifuge tube and shakes mixing; Instantaneous centrifugal after; Packing 45 μ l in each PCR reaction tubes transfer to sample application zone behind the lid upper tube cap, and it is subsequent use that lucifuge is put 4 ℃ of refrigerators.
3. reference substance and quantitative reference article are transferred to the sample preparation district, be put in 4 ℃ of refrigerators subsequent use.
Two, sample process (sample process district)
1. be suitable for the sample type: reproductive tract, urethral secretions swab etc.
2. collection of specimens:
2.1 male urethra sample: insert the rotation of the about 2cm of urethra place with sterile swab, obtain the secretory product sample after the static several seconds, put back to airtight censorship in the pipe.
2.2 female urethra sample: clean urethral orifice with SPSS earlier, insert the about 2cm of urethra place's rotation with sterile swab again and obtain the secretory product sample, put back to airtight censorship in the pipe.
2.3 female genital tract sample: earlier wipe the too much secretory product of intravaginal with SPSS, again with sterile swab anti-place reproductive tract low level 1/3 place gently rotation obtain mucous membrane secretory product sample, put back to airtight censorship in the pipe.
3. sample is preserved and censorship: sample can be inspected by ready samples at once, should not surpass 6 days 4-25 ℃ of preservation, and on-liquid property sample also can be preserved 6 months at-20 ℃.Should use ice bag during the long-distance censorship of sample.
4 samples to be tested are handled:
4.1 in the swab pipe, add the 1ml sterile saline, shake 1min at a high speed, extract swab;
4.2 the whole liquid after handling 1.1 are transferred in the 1.5ml centrifuge tube and (like excess secretion, are only got 200 μ l), centrifugal 5 minutes of 10000rpm;
4.3 remove supernatant, in deposition, add the 1ml sterile saline, abundant mixing, centrifugal 5 minutes of 10000rpm;
4.4 remove supernatant, in deposition, add 50 μ lDNA extracting solutions (extracting solution includes water-fast material, fully draws behind the mixing), abundant mixing, 100 ℃ of constant temperature were handled 10 minutes;
1.510000rpm centrifugal 5 minutes, supernatant was used for the PCR reaction.
5. negative quality control product sample process
5.1. take out negative quality control product, instantaneous centrifugal, get 50 μ l in the 1.5ml centrifuge tube, add 50 μ lDNA extracting solutions (extracting solution includes water-fast material, fully draws behind the mixing), abundant mixing, 100 ℃ of constant temperature were handled 10 minutes;
5.2. instantaneous centrifugal, supernatant is used for the PCR reaction.
6. positive quality control product sample process: (with negative quality control product).
7. weak positive quality control product sample process: (with negative quality control product).
8. quantitatively the reference article are handled: the centrifugal several seconds of 10000rpm, subsequent use.
Three, PCR reaction
1, application of sample (sample preparation district or sample application zone)
In ready PCR reaction solution pipe, add testing sample, negative quality control product sample, positive quality control product, the weak positive quality control product sample of handling well, supernatant 5 μ l respectively.Or directly adding quantitative reference article 5 μ l, the tight pipe of lid covers the instantaneous low-speed centrifugal in back.
2, pcr amplification (detection zone)
Ready PCR reaction tubes is positioned on the PCR appearance, edits sample information and carry out amplified reaction by table 3 loop parameter:
Table 3
Comprise in the UU-PCR reaction system: UU detects and internal reference.
Reference value (term of reference) utilizes the instrument software kit to analyze automatically like table 3, obtains each sample Ct value and C value.
Table 4
Four, interpretation of result
A, condition enactment:
1.ABI 7500 baselines (baseline) are set: get 2 and arrive preceding 3 cycle values of sample threshold value (threshold) as baseline.Threshold value (threshold) setting principle is with the vertex of threshold line just above normal negative quality control product amplification curve (random noise line), promptly the negative quality control product of Ct=40 perhaps " Undet. " be as the criterion.
2.STRATAGENE the Mx3000P baseline is set: select " being fit to baseline (Adaptive baseline) " to establish periodic fluorescent signal.Threshold value (threshold) setting principle is with the vertex of threshold line just above normal negative quality control product amplification curve (random noise line), promptly the negative quality control product of Ct=40 perhaps " No Ct " be as the criterion.
B, quality control standard:
This test kit positive and negative quality control product should meet the following conditions simultaneously, otherwise it is invalid to be regarded as this experiment:
1. negative quality control product: UU (FAM) Ct value=40 or " No Ct " be " Undet. " (ABI 7500) (Mx3000P) perhaps, internal reference (HEX) Ct value＜40, and logarithmic growth curve is preferably arranged.
2. positive quality control product: UU (FAM) has logarithmic growth curve preferably.Quantitatively reference value is 1.0 * 10
6Copies/ml-1.0 * 10
7Copies/ml.
3. weak positive quality control product: UU (FAM) has logarithmic growth curve preferably.Quantitatively reference value is 1.0 * 10
3Copies/ml-1.0 * 10
4Copies/ml.
C, result judge
1.UUCt value=40 or " No Ct " be " Undet. " (ABI 7500) (Mx3000P) perhaps; And internal reference (HEX) channel C t value＜40, and logarithmic growth curve is preferably arranged, then the dna content of UU is less than detecting lower limit.
2.UUCt value＜40, and logarithmic growth curve is preferably arranged, then by following method interpretation:
2.1 sample C＜5.00E+002, the dna content of UU=C gene copy (only for reference, recheck, if still have logarithmic growth curve preferably, then positive by suggestion.)；
2.2 sample 5.00E+002≤C≤5.00E+008, the then dna content of UU=C gene copy;
2.3 the dna content of sample UU＞5.0 * 10
8Copies/ml quantitatively can be diluted to sample in the linearity range like need and to redeterminate.
3.UUCt value=40 or " No Ct " be " Undet. " (ABI 7500) (Mx3000P) perhaps; And internal reference (HEX) Ct value=40 or " No Ct " (Mx3000P) perhaps " Undet. " (ABI7500) increase and fail, and please redeterminate.
See also Fig. 1, Fig. 2, Fig. 3, Fig. 4,, show that it possesses following performance through this product accuracy, specificity, repeatability, stability, sensitivity and precision are studied:
Fig. 1 detects figure as a result for sensitivity reference article, and the concentration of surveying is followed successively by 5.0 * 10
4Copies/ml, 5.0 * 10
3Copies/ml, 5.0 * 10
2Copies/ml, 5.0 * 10copies/ml.Minimum detectability is 5.0 * 10
2Copies/mL.
Fig. 2: typical curve reference article detect figure as a result, are followed successively by 1.0 * 10
7Copies/ml, 1.0 * 10
6Copies/ml, 1.0 * 10
5Copies/ml, 1.0 * 10
4Copies/ml Fig. 3 is the typical curve made from Fig. 2 result.
Fig. 4: the UU specific detection is figure as a result, is followed successively by: T1: Candida albicans, T2: streptococcus aureus, T3:B family suis, T4: chlamydia psittaci, T5: CPN, T6: chlamydia trachomatis, T7: mycoplasma genitalium, T8: mycoplasma hominis, T9: intestinal bacteria, T10: staphylococcus epidermidis.And positive control, weak positive control.It is all negative to detect the internal control reference article T1-T10 of enterprise sample results.
To sum up, compare with laboratory regular-PCR detection by quantitative, this product accuracy is high, high specificity, and in repeatability, stability, sensitivity and precision very big improvement has been arranged all, improves, and keeping life is 6 months, and product performance are stable before the deadline.
This test kit has 5 tangible characteristics:
1, DNA extraction is effective;
2, have very high accuracy, sensitivity and specificity;
3, need not the PCR aftertreatment, the dUTP-UNG system has been adopted in stopped pipe amplification and detecting fully, helps avoid the pollution of amplified production;
4, weak point simple to operate, consuming time (2～3 hours);
5, the result is objective reliable, the automatic Collection and analysis data of instrument.
The above is merely embodiments of the invention; Be not so limit claim of the present invention; Every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to be done; Or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Claims (7)
1. a ureaplasma urealyticum PCR detection kit comprises the PCR reaction solution, it is characterized in that, said PCR reaction solution comprises primer and fluorescent probe A, and said primer is divided into upstream primer A and downstream primer A;
The nucleotides sequence of said upstream primer A is classified as: 5 '-GCTAAATCAACAGACGGT-3 ';
The nucleotides sequence of said downstream primer A is classified as: 5 '-GCTGCACTTACATCAGCTG-3 ';
The nucleotides sequence of said fluorescent probe A is classified as:
5’-TGCGGTTTACGAAATTGAAAACT-3’。
2. by the described ureaplasma urealyticum PCR of claim 1 detection kit, it is characterized in that, also comprise DNA extraction liquid, dNTPs, tap enzyme, UNG enzyme, positive control, negative control, quantitative reference article.
3. by the described ureaplasma urealyticum PCR of claim 2 detection kit, it is characterized in that said dNTPs comprises dATP, dCTP, dGTP, dCTP.
4. by the described ureaplasma urealyticum PCR of claim 1 detection kit; It is characterized in that; Having introduced an artificial constructed internal reference system is used to avoid the false-negative internal reference of detected result system to comprise confidential reference items probe, primer and confidential reference items DNA; Said internal reference system is an Arabidopis thaliana internal reference system, and said internal reference system comprises upstream primer B:SEQ ID NO.5, downstream primer B:SEQ ID NO.6, fluorescent probe: SEQ ID NO.7, specific as follows:
Upstream primer B:5 '-TCATCGTCGCTGGAGCTGGTT-3 '
Downstream primer B:5 '-CGGCGGTTTGTCAAGCTGAT-3 '
Fluorescent probe B:
5′-HEX-CTTCTTATAGTCACTGCACTAAACTGGAT-TAMRA-3′。
5. by the described ureaplasma urealyticum PCR of claim 1 detection kit, it is characterized in that 5 ' end flag F AM fluorophor of said fluorescent probe, 3 ' end mark BHQ quenching group.
6. ureaplasma urealyticum PCR detection method; It is characterized in that wherein the target sequence of pcr amplification is SEQ ID NO.4, the pcr amplification the primer is: upstream primer A sequence is like SEQ ID NO.1; Downstream primer A sequence is like SEQ ID NO.2, and fluorescent probe A sequence is like SEQ ID NO.3.
7. by the described ureaplasma urealyticum PCR of claim 6 detection method, it is characterized in that 5 ' the end flag F AM fluorophor of said fluorescent probe A, 3 ' end mark TAMRA quenching group.
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Cited By (4)
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| 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 |
| CN107557456A (en) * | 2017-09-29 | 2018-01-09 | 广州洪祥生物医药科技有限公司 | The LAMP detection primer group and kit of ureaplasma urealyticum |
| CN110257534A (en) * | 2019-04-22 | 2019-09-20 | 深圳市国赛生物技术有限公司 | Ureaplasma urealyticum kit for detecting nucleic acid |
| 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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| CN101613763A (en) * | 2009-07-30 | 2009-12-30 | 港龙生物科技有限公司 | The fluorescence PCR method of diagnosis chlamydia trachomatis, gonococcus and ureaplasma urealyticum infection |
| CN102102121A (en) * | 2009-12-17 | 2011-06-22 | 上海裕隆临床检验中心有限公司 | Fluorescent PCR kit for detecting ureaplasma urealyticum |
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| 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 |
| CN103074429B (en) * | 2013-01-10 | 2014-09-24 | 湖南圣湘生物科技有限公司 | UU (ureaplasma urealyticum) detection kit |
| CN107557456A (en) * | 2017-09-29 | 2018-01-09 | 广州洪祥生物医药科技有限公司 | The LAMP detection primer group and kit of ureaplasma urealyticum |
| CN107557456B (en) * | 2017-09-29 | 2020-06-16 | 广州洪祥生物医药科技有限公司 | LAMP (loop-mediated isothermal amplification) detection primer group and kit for ureaplasma urealyticum |
| CN110257534A (en) * | 2019-04-22 | 2019-09-20 | 深圳市国赛生物技术有限公司 | Ureaplasma urealyticum kit for detecting nucleic acid |
| 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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