CN112410442A - Method and kit for detecting ureaplasma urealyticum - Google Patents
Method and kit for detecting ureaplasma urealyticum Download PDFInfo
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Abstract
The invention provides a detection method and a kit for ureaplasma urealyticum, wherein the detection method comprises the following steps: (1) extracting DNA of a sample and determining the concentration of the DNA; (2) after dilution, the copy number of the gene of interest uif was detected using ddPCR. The detection kit comprises primers of SEQ ID NO.1 and SEQ ID NO.2 and a probe of SEQ ID NO.3, wherein the probe is marked with fluorescence. The detection kit and the detection method for ureaplasma urealyticum have the advantages of good specificity, high detectable rate, less false positive and capability of absolute quantitative analysis.
Description
Technical Field
The invention relates to microbial detection, in particular to a method and a kit for detecting ureaplasma urealyticum.
Background
Ureaplasma Urealyticum (UU) belongs to the mollicutes, Ureaplasma ( serotypes 2, 4, 5 and 7-13), is a kind of minimal prokaryotic cell type microorganism which has no cell wall, is highly polymorphic, can pass through a sterilizing filter and can grow and reproduce in an inanimate culture medium [1], is one of the common colonized microorganisms in the urogenital tract of women, and the proportion of the vertical transmission of the UU of a newborn is reported to be 45-66%, and is higher than that of a premature infant.
UU can cause intrauterine infection of the perinatal infants through the ways of ascending infection of the genital tract, blood circulation infection through placenta and the like, and lead to poor pregnancy fatalities such as premature birth, intrauterine growth retardation, intrauterine pneumonia, bronchopulmonary dysplasia, perinatal death and the like. Many premature births of unknown origin are caused by UU infection. Studies by Ho Seon Eun et al reported that UU infection in premature infants is associated with BPD, especially with the formation of moderately severe BPD [3-4 ]. Therefore, the detection rate of UU is improved, the copy number level change of UU nucleic acid of a diagnosed child after treatment is improved, and a clinical theoretical basis is provided for understanding the relationship between UU positivity and BPD of a premature child and evaluating the treatment effect of UU infection. In view of the above situation, there is a need to develop a new detection technology for UU pathogenic bacteria, which is one of the problems to be solved urgently.
At present, UU is mainly detected by a culture method and a fluorescent quantitative PCR (qPCR) method. Microbial culture identification is the gold standard for UU detection. However, the false negative rate of microbial culture is high, and the culture period is long (5-7 days), so that the UU is generally detected by using a fluorescent quantitative PCR method at present. Although the qPCR detection time is obviously shortened compared with the microbial culture method, the original Ct value of the sample in the qPCR must depend on external reference to establish a standard curve, so the reliability and repeatability of the standard substance seriously affect the repeatability and reliability of the quantitative result of an unknown sample, for example, the standard substance can cause the concentration of a sample to be detected to be seriously deviated due to degradation in different preparation processes or storage processes; in addition, quantitative analysis can be carried out only by means of a standard curve when an unknown sample is detected by qPCR, when the concentration of a template is extremely low, the amplification efficiency of the qPCR is reduced due to the reduction of the binding probability of primers, probes and polymerase with a target sequence of the template in the qPCR reaction, so that the Ct of the commercial qPCR clinical detection kit is less than or equal to 30 (about 1000copies/20 mul reaction system) as a positive result, and the detection rate of pathogenic bacteria is greatly reduced; meanwhile, impurities such as bilirubin and salts in a sample or detection system can affect the detection result.
In the droplet digital PCR (ddPCR), a sample is subjected to a microdroplet treatment before conventional PCR amplification, that is, a reaction System containing nucleic acid molecules is divided into thousands of nanoliter droplets, wherein each droplet contains no nucleic acid target molecules to be detected or contains one to several nucleic acid target molecules to be detected. After PCR amplification, the microdroplets containing the nucleic acid target molecules to be detected generate fluorescent signals, and the microdroplets not containing the nucleic acid target molecules to be detected do not generate fluorescent signals. And finally, calculating the initial concentration or copy number of the nucleic acid target molecules to be detected according to the Poisson distribution principle and the proportion of the positive microdroplets.
Compared with the traditional PCR, ddPCR has the advantage that the absolute number of target molecules to be detected can be determined as low as a single copy without depending on Ct value or internal reference genes. The microdroplet technology makes digital PCR more cost effective and practical, and ddPCR eliminates the dependence on quantitative standard curve and improves the tolerance to amplification inhibitor.
Disclosure of Invention
In view of the above problems, an object of the present invention is to provide a detection kit for ureaplasma urealyticum, which has high specificity and high detectable rate and can be used for quantitative analysis.
A detection kit for ureaplasma urealyticum comprises primers and probes for uif genes, wherein the primers are SEQ ID NO.1 and SEQ ID NO.2, the probes are SEQ ID NO.3, and the probes are marked with fluorescence.
In some embodiments, the kit further comprises a negative quality control, wherein the negative quality control is sterilized normal saline.
In some of these embodiments, a positive control is also included, the positive control being a ureaplasma urealyticum cDNA template.
In some embodiments, the digital PCR reaction buffer, hot start Taq enzyme and UNG enzyme are also included.
Another object of the present invention is to provide a method for detecting ureaplasma urealyticum for non-diagnostic purposes.
Based on the above purpose, the invention provides the following technical scheme:
a method for detecting ureaplasma urealyticum for non-diagnostic purposes, comprising the steps of:
(1) extracting DNA in a sample and determining the concentration thereof;
(2) the copy number of the ureaplasma urealyticum uif gene in the sample is detected by ddPCR by using the kit.
In some of the embodiments, the annealing temperature of the ddPCR is 55-59 ℃, and more preferably 55 ℃.
In some embodiments, the probe has a final concentration of 0.2-0.3 mmol/L, and more preferably 0.25mmol/L in the reaction system of ddPCR.
In some embodiments, the final concentration of the primers SEQ ID NO.1 and SEQ ID NO.2 in the reaction system of ddPCR is 0.9-1.1 mmol/L, and more preferably 1 mmol/L.
In some of these embodiments, the sample of step (1) comprises pharyngeal swab secretions.
Based on the technical scheme, the invention has the following beneficial effects:
the invention designs and obtains a primer and a probe sequence with excellent detection effect by aiming at the conserved uif gene in the ureaplasma urealyticum, is used for detecting the ureaplasma urealyticum in a sample by a ddPCR method, has the lower detection limit of 5copies/20 mu L, has good specificity and sensitivity, obviously shortens the detection time compared with a microbial culture method, and is suitable for detecting various types of samples.
The ureaplasma urealyticum detection kit is not influenced by impurities in a standard substance and a detection sample, and compared with a qPCR method, the repeatability of the detection method is obviously improved, and the detection product has a wide application prospect in clinic.
Drawings
FIG. 1 is a diagram showing the detection results of the probe and the primer for detecting UU by ddPCR, wherein (a) is a diagram showing the optimized scatter diagram of the annealing temperature of ddPCR, and (b) is a diagram showing the detection results of the specificity of UU by ddPCR.
FIG. 2 shows the result of ddPCR detection of UU DNA by linear regression analysis.
FIG. 3 shows the detection of UU DNA with low copy number by ddPCR.
Detailed Description
The invention provides a method for detecting ureaplasma urealyticum. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations. The various reagents used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
1.1 materials
1.1.1 Experimental materials
Ureaplasma Urealyticum (UU) available from the china culture collection (CMCC); mycoplasma hominis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Escherichia coli, group B streptococcus, Klebsiella pneumoniae, Candida albicans, and Candida glabrata were provided by the department of gynaecological healthcare institute, Shengzhen, Nostoc.
1.1.2 instruments
A NanoDrop Lite uv spectrophotometer (seimer feishell science ltd, usa); QX200 Droplet digital PCR system (including PCR instrument, QX200 Droplet Generator, PX1 Plate Sealer and QX200 Droplet Reader) (Bio-rad, USA); 7500 fluorescent quantitative PCR instrument (Applied Biosystems, USA).
1.1.3 reagents
Tryptone soy broth, blood agar base purchased from Haibobo; aseptic defibered sheep blood was purchased from yikang biology; lambda-Hind III digest, DL 200DNAmarker, restriction enzyme, Premix TaqTMHot Start Version, Takara MiniBEST Plasmid Purification Kit, Takara Agarose Gel DNA Purification Kit and so on from TaKaRa corporation, Japan; clinical sample DNA extraction kit
DNAmii Kit was purchased from Qiagen, GermanyA driver; q-PCR kit iTaqTMUniversal Probes Supermix, ddPCR kit 2 × ddPCR Supermix (ddPCR reaction buffer, Hot Start Taq enzyme and UNG enzyme), microdroplet oil; a droplet generation card; 96-well PCR plates; the gasket was purchased from Bio-Rad, USA; gene sequencing was performed by Guangzhou Egyki Biotechnology, Inc.
1.2 methods
1.2.1UU Mycoplasma Strain identification
UU is placed in a mycoplasma broth containing urea, and a phenol red indicator is added, NH3 is generated after urea is decomposed by the UU, and the mycoplasma broth changes color to indicate that the UU is positive.
1.2.2 suspension preparation
Activating the UU transfer agar solid culture medium after enrichment. Circular brown colonies appeared 2-3d after inoculation on agar plates. Selecting a single UU colony to perform incubation on a solid agar culture medium
Selecting a plate in an effective range of 30-300 CFU by adopting a plate colony counting method, counting the number of colonies, and calculating the original concentration of the bacterial liquid according to the following formula:
original concentration of bacteria (CFUs/mL) ═ average colony count (CFUs. times. dilution factor)/0.1 mL
1.2.3 extraction and identification of UU Strain DNA
And collecting bacterial liquid and extracting whole genome DNA.
Negative quality control is established with sterilized normal saline.
Urea ureaplasma urealyticum cDNA was used as a positive control.
1.2.4 establishment of UU-ddPCR method
Specific primers and probes capable of amplifying uif1 and uif2 respectively are designed according to nucleotide sequences of the UU conserved gene uif, and the nucleotide sequences of the primers and the probes are shown in Table 1. The detection is carried out by a ddPCR method, and the result shows that the specificity of the primer and the probe designed by adopting the uif1 sequence is superior to uif2, so that the primers adopted in the UU-ddPCR method established by the invention are SEQ ID NO.1 and SEQ ID NO.2, the probe is SEQ ID NO.3, and the probe is marked by a fluorescent group.
TABLE 1 uif primer and Probe sequences
The ddPCR reaction condition is 95 ℃ for 5 min; 10sec at 98 ℃, 30sec at 55 ℃, 10sec at 72 ℃ for 45 cycles; 10min at 72 ℃. And (3) carrying out electrophoresis on the PCR product by using a 1% agarose gel and sequencing for identification.
20 μ L ddPCR amplification system (20 μ L):
2×ddPCR Supermix(10μL)；
10mmol/L primer mixture (2. mu.L each);
10mmol/L probe (0.5. mu.L);
UU strain DNA (5. mu.L);
sterilized deionized water (0.5. mu.L).
1.2.5UU-ddPCR amplification Condition optimization
The annealing temperature affects the specificity of PCR and is an important parameter of PCR reaction conditions, the sensitivity of the reaction is reduced when the annealing temperature is too high, and nonspecific amplification can be caused when the annealing temperature is too low. The ddPCR reaction program was determined by optimizing the annealing temperature.
The reaction program is as follows:
the temperature rise and fall speed is set to be 2 ℃/s for each temperature.
The annealing temperature gradients are respectively 8 gradients of 60 ℃, 59.4 ℃, 58.3 ℃, 55 ℃, 53.9 ℃, 52 ℃, 50.7 ℃ and 50 ℃, and UU DNA is used as a template to carry out experiments to determine the optimal annealing temperature.
20 μ L ddPCR amplification system (20 μ L):
2×ddPCR Supermix(10μL)；
10mmol/L primer mixture (2. mu.L each);
10mmol/L probe (0.5. mu.L);
UU Strain DNA (5. mu.L)
Sterilizing deionized water (0.5 mu L)
1.2.6ddPCR detection of copy number of UU COH1 Strain uif
And ensuring that the concentration of the bacterial liquid for extracting DNA each time is basically consistent according to a colony counting method, and measuring the concentration by an ultraviolet spectrophotometer after extracting the DNA. According to the following formula, the estimated concentration is diluted by 10 times and then subjected to ddPCR detection, 3 subplates are detected in each subpell, the template amount detected in each subpell is 5 mul, and the experiment is repeated three times.
C2＝(NA×C1×10-9)/(L×660)
C1To estimate the concentration, C2For the DNA concentration detected by spectrophotometer (copies/. mu.L), L is the genome length (UU genome length about 23367 bp).
1.2.7 detection Range and lower detection Limit of UU-ddPCR
Will know the concentration 106copies/. mu.LUU DNA was diluted to 10 with enzyme-free water and a gradient containing normal human pharyngeal secretions (UU infection was verified by plating and dPCR) respectively5-101copies/μL。
The lower detection limit of UU-ddPCR was evaluated by diluting 10 copies/. mu.L, 5 copies/. mu.L, 2.5 copies/. mu.L of 10 copies/. mu.L DNA with enzyme-free water and DNA containing normal human pharyngeal secretions (the absence of UU infection has been verified by plating and dPCR), respectively, at a double ratio.
1.2.8 specimen source and collection method
The specimen is a premature infant which is born in obstetrics of maternal and child healthcare institutions in Shenzhen city from 3 months to 9 months in 2018, has gestational age less than or equal to 32 weeks and is in a neonatal intensive care unit of the Shenzhen city. Exclusion criteria: the premature infant is treated by a transfer operation of surgical complications such as definite severe bacterial infection or early septicemia when the newborn is born, severe asphyxia when the newborn is born, hereditary or metabolic diseases of mother or newborn, severe congenital malformation when the newborn is born, intestinal perforation and the like.
Pharyngeal swab specimens were left in dry sterile tubes within 2 hours after the preterm infant. And (3) putting the swab head into 1ml of normal saline, soaking the swab head, sticking the swab head to the wall of the tube, squeezing the swab head to be dry, taking 0.5ml, adding 0.5ml of sample preservation solution, and mixing to obtain the sample to be detected.
1.2.8UU-ddPCR and UU-qPCR detection sensitivity, specificity analysis.
The collected clinical specimen is divided into 2 parts, 1 part is identified by a culture method, 1 part is extracted with nucleic acid DNA (the purity OD260/280 is ensured to be 1.7-1.9, the concentration is 25-100 ng/mu L), and Real-time qPCR and ddPCR are respectively carried out for detection.
1.2.9 diagnostic standard and related index of UU:
bronchopulmonary dysplasia (BPD): any oxygen dependent (> 21%) preterm infant with 21 oxygen days. Clinical graduation: graduations were made according to corrected gestational age at 36 weeks or discharge aerobic concentration: the method is mild: no oxygen is used; moderate degree: inhalation oxygen concentration (Fi02) < 30%; ③ the degree of gravity: fi02 degrees: oxygen% or mechanical aeration is required. Pulmonary x-ray representation is not used as a basis for assessment of disease severity.
Ureaplasma Urealyticum (UU) positive: a throat swab specimen is taken within 2 hours after the premature infant, UU-RNA is detected by a Polymerase Chain Reaction (PCR) method, and the result is positive, namely that the UU is positive.
Patent Ductus Arteriosus (PDA): patent ductus arteriosus is said to be a patent ductus arteriosus which is continuously open in the presence of factors such as poor ductus wall development, abnormal secretion of prostaglandins, etc. after the premature infant.
Neonatal Respiratory Distress Syndrome (RDS): after the birth of the neonate, progressive dyspnea, cyanosis and respiratory failure occur for hours due to the lack of lung surfactant.
Periventricular-intracerebroventricular hemorrhage (IVH) of premature infants: when the ventricular membrane of the premature infant is ruptured, i.e. the ventricular subintimal hemorrhage, the ventricular subintimal hemorrhage is formed when the ventricular membrane is ruptured and blood flows into the ventricles, which is called the ventricular subintimal hemorrhage of the premature infant. The imaging examination is divided into I degree, II degree, III degree and IV degree according to the bleeding occurrence and development process, bleeding amount and bleeding degree.
Retinopathy of prematurity (ROP): premature retinal blood vessels are immature, and during further maturation of blood vessels, the retina is hypoxic due to increased metabolic demand, retinal vasoconstriction, occlusion, etc., stimulating neovascularization, and eventually leading to ROP.
Prenatal dexamethasone: preterm infants who used 5mg dexamethasone for q12hx2 days prior to delivery of the pregnant woman.
Invasive assisted ventilation: the trachea cannula is connected with an invasive respirator to assist the ventilation of the premature infant after the birth.
1.2.10ddPCR and clinical outcome-related analysis
Collecting the data of the birth condition of the premature infant, including birth weight, gestation period, sex, delivery mode, premature rupture time of fetal membranes, prenatal dexamethasone application and the like. The incidence of complications of premature infants including bronchopulmonary dysplasia (BPD), Patent Ductus Arteriosus (PDA), periventricular-intraventricular hemorrhage (IVH) of premature infants, pulmonary hyaline membrane disease (RDS) of newborn infants, retinopathy of prematurity (ROP), etc. was collected. Collecting data related to diagnosis and treatment processes of the premature infant during the hospitalization period, including the use condition of the premature infant, the presence or absence of invasive auxiliary ventilation, the oxygen consumption time, the hospitalization time and the like.
1.2.11 statistical analysis
Statistical analysis software SPSS 21.0 was used for data analysis. The sensitivity and specificity of qPCR and ddPCR methods in UU detection are tested by adopting chi-square method; independent risk factors for analyzing whether the independent variables are dependent variables or not adopt multifactor logistic regression. P is less than 0.05, which has statistical significance.
2 results and analysis
2.1UU-ddPCR assay specificity evaluation
In order to determine the optimal annealing temperature of the UU-ddPCR established by the invention, the annealing temperatures are respectively set to be 60 ℃, 59.4 ℃, 58.3 ℃, 55 ℃, 53.9 ℃, 52 ℃, 50.7 ℃ and 50 ℃ with 8 gradients. The reaction result is shown in fig. 1(a), the most suitable annealing temperature range is 55-59 ℃, under the temperature range, the positive fluorescent droplet and the negative droplet are separated most, 55 ℃ is selected as the annealing temperature, and the effect is best at the temperature.
In order to determine the specificity of the UU-ddPCR established by the invention, 8 pathogenic microorganisms (table 2) and UU with high perinatal infection rate are selected for detection, DNA of a strain to be tested is used as a detection template, and the detection result shows that only the UU sample has positive microdroplets (figure 1(b)) and other pathogenic microorganisms do not have positive microdroplets, so that the UU-ddPCR detection method established by the invention has high specificity.
TABLE 2UU-ddPCR detection of the specificity of the genes of interest
| Sample(s) | Bacterial | ddPCR results | |
| 1 | Ureaplasma urealyticum | + | |
| 2 | Mycoplasma hominis | - | |
| 3 | Mycoplasma pneumoniae | - | |
| 4 | Mycoplasma genitalium | - | |
| 5 | Chlamydia trachomatis | - | |
| 6 | Water (blank control) | - |
2.2 detection Range of UU-ddPCR
The UU DNA was diluted in a 10-fold gradient and subjected to ddPCR detection and linear analysis of the detection results (fig. 2). The results show that: GBS-ddPCR is 10-105In the range of copies/20. mu.L R20.9968. Show that the ratio is 10 to 105Within the dynamic range of copies/20 mu L, the ddPCR detection method has better linear relation.
2.3 determination of lower limit of detection of clinical samples for UU-ddPCR
Human clinical specimens were simulated by diluting 10 copies/. mu.L UU DNA to 10 copies/. mu.L, 5 copies/. mu.L and 2.5 copies/. mu.L, respectively. Dilutions were enzyme-free and DNA containing normal human vaginal perianal secretion (UU infection was verified by plating and dPCR) and 5 replicates were performed. The results are shown in FIG. 3. The sensitivity of ddPCR of the sample containing human DNA is 5copies/20 mu L, and the lower detection limit of the sample without human DNA can reach 2.5copy/20 mu L.
2.4 to evaluate the consistency of UU-qPCR, UU-ddPCR and the microbial culture method, chi-square test was performed on 53 cases of unknown sample data, and the results showed (Table 4) that UU-qPCR was detected with sensitivity of 90.0% (66.9% -98.2%) and specificity of 90.9% (74.5% -97.6%). UU-ddPCR showed 95.0% detection sensitivity (73.1% -99.7%) and 93.9(78.4-98.9) specificity.
TABLE 3 analysis of the results of the culture method, qPCR and ddPCR
2.5UU-ddPCR primer and probe in the preparation of clinical diagnosis premature infant UU infection kit
According to the identification of clinical outcomes such as BPD in the UU positive premature infant and the UU negative group by ddPCR, the difference of oxygen time and bronchopulmonary dysplasia (BPD) has statistical significance, P is less than 0.05, the oxygen time of the UU positive group is longer, and the BPD prevalence rate is higher. Comparing the hospitalization time and invasive auxiliary ventilation rate of the two groups, wherein P is more than 0.05, and the difference has no statistical significance; comparing the incidence of other complications of premature infants in both groups, such as Patent Ductus Arteriosus (PDA), periventricular-intraventricular hemorrhage (IVH) of premature infants, pulmonary hyaline disease (RDS) of newborn, retinopathy of prematurity (ROP), P is > 0.05, the difference is not statistically significant.
The results are shown in Table 4.
TABLE 4 comparison of clinical outcomes of UU-positive and UU-negative groups
The above study results show that: the lower limit of the UU-ddPCR detection method established by the invention is 5copies/20 mu L, and the method is not influenced by impurities in a sample, so that the sensitivity and the repeatability of the detection method are ensured. Meanwhile, the sensitivity of UU-ddPCR established by the invention to secretion detection results is 95.0% (73.1% -99.7%), and the specificity is 93.9% (78.4% -98.9%), which are slightly higher than those of the traditional qPCR detection method. In addition, the sensitivity and the repeatability of the UU-ddPCR established by the invention are superior to those of qPCR, compared with a microbial culture method, the detection time is obviously shortened, and the method is suitable for detection of different clinical samples. The UU-ddPCR established by the invention is used for comparing the clinical outcomes of BPD and the like in the UU positive group and the UU negative group, the difference of oxygen time and bronchopulmonary dysplasia (BPD) has statistical significance, P is less than 0.05, the oxygen time of the UU positive group is longer, and the BPD morbidity is higher.
The technical features of the above-mentioned embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the following embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the combinations should be considered as the scope of the present description.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (10)
1. A detection kit for ureaplasma urealyticum is characterized by comprising primers and probes for uif gene, wherein the primers are SEQ ID NO.1 and SEQ ID NO.2, the probes are SEQ ID NO.3, and the probes are marked with fluorescence.
2. The ureaplasma urealyticum detection kit according to claim 1, further comprising a negative quality control, wherein the negative quality control is sterilized normal saline.
3. The ureaplasma urealyticum detection kit according to claim 1, further comprising a positive control, wherein the positive control is a ureaplasma urealyticum cDNA template.
4. The ureaplasma urealyticum detection kit according to any one of claims 1 to 3, further comprising ddPCR reaction buffer, hot start Taq enzyme and UNG enzyme.
5. A method for detecting ureaplasma urealyticum for non-diagnostic purposes, comprising the steps of:
(1) extracting DNA in a sample and determining the concentration thereof;
(2) the use of the kit of any one of claims 1 to 4 for ddPCR detection of the copy number of ureaplasma urealyticum uif gene in a sample.
6. The method for detecting ureaplasma urealyticum for non-diagnostic purposes according to claim 5, wherein the annealing temperature of ddPCR is 55 to 59 ℃.
7. The method for detecting ureaplasma urealyticum for non-diagnostic purposes as claimed in claim 6, wherein the annealing temperature of ddPCR is 55 ℃.
8. The method for detecting ureaplasma urealyticum for non-diagnostic purposes according to claim 5, wherein the final concentration of the probe in the reaction system of ddPCR is 0.2 to 0.3mmol/L, and/or the final concentrations of the primers SEQ ID No.1 and SEQ ID No.2 in the reaction system of ddPCR are 0.9 to 1.1mmol/L, respectively.
9. The method for detecting ureaplasma urealyticum for non-diagnostic purposes as claimed in claim 8, wherein the probe has a final concentration of 0.25mmol/L in the reaction system of ddPCR, and/or the primers SEQ ID NO.1 and SEQ ID NO.2 have a final concentration of 1mmol/L in the reaction system of ddPCR, respectively.
10. The method for detecting ureaplasma urealyticum for non-diagnostic purposes according to any one of claims 5 to 8, wherein the sample in step (1) is a throat swab secretion.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113684287A (en) * | 2021-08-31 | 2021-11-23 | 中国科学院上海微系统与信息技术研究所 | Primer probe group, PCR reagent and kit for detecting mycoplasma pneumoniae |
| 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113684287A (en) * | 2021-08-31 | 2021-11-23 | 中国科学院上海微系统与信息技术研究所 | Primer probe group, PCR reagent and kit for detecting mycoplasma pneumoniae |
| 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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