CN112458194B - Primer probe combination and detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof - Google Patents
Primer probe combination and detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention relates to the technical field of biology, in particular to a primer probe combination and a detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof. The primer probe combination comprises: as set forth in SEQ ID NO: 1-2, a specific primer pair of mycoplasma pneumoniae; as set forth in SEQ ID NO:3, a mycoplasma pneumoniae-sensitive detection probe; as set forth in SEQ ID NO:4, detecting a drug-resistant mutation of a mycoplasma pneumoniae 2063 site; and as set forth in SEQ ID NO:5, and detecting the drug-resistant mutation of the position 2064 of mycoplasma pneumoniae. The primer and the probe have good specificity, and can realize quick, accurate and sensitive identification and calculation of drug resistance proportion of mycoplasma pneumoniae and drug resistance mutation thereof by combining with a real time PCR detection method.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer probe combination and a detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof.
Background
Mycoplasma Pneumoniae (MP) is an important pathogen in humans and can cause community infections. In recent years, with the widespread use of antibiotics, some mycoplasma pneumoniae has drug resistance to tetracycline antibiotics, and the phenomenon of drug resistance enhancement is becoming more and more important.
The detection method of mycoplasma pneumoniae is various, the separation culture is a 'gold standard' for microorganism detection, the method is reliable, but the method is time-consuming and labor-consuming, the requirement of rapid detection cannot be met, and the common immunofluorescence method has certain specificity and sensitivity, but the result is unstable due to the influence of the type and concentration of antibodies, and false positives can sometimes occur. In order to overcome the problems, the multiplex fluorescence quantitative RT-PCR technology is used for diagnosing mycoplasma pneumoniae and drug-resistant infections thereof, the method has the characteristics of rapidness, sensitivity, strong specificity and the like, and the probe can distinguish the sensitivity type from the drug-resistant type and calculate the proportion of the drug-resistant bacteria, thereby greatly improving the detection efficiency.
However, at present, the design of primer probes for mycoplasma pneumoniae and drug resistance identification thereof is unreasonable, so that most detection reagents cannot realize accurate, rapid and sensitive detection. The detection sensitivity of the primer probe according to the invention of the authorized publication No. CN 102002522B is 100Copies/ml. The detection sensitivity of the primer probe disclosed by the publication No. CN 106191239A is 1000Copies/ml, 3 tubes are needed for detection in the detection process, the workload is large, and the detection flux is low. The primer probe disclosed by the publication No. CN 103820557A can only detect sensitive strains and 2063 site mutant strains. And the drug resistance ratio of mycoplasma pneumoniae mixed infection cannot be judged by the three patent methods.
Disclosure of Invention
In view of the above, the invention provides a primer probe combination and a detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof. The minimum detection limit of the primer probe combination on mycoplasma pneumoniae sensitivity and mutant type is 50copies/mL, and the drug resistance ratio of mycoplasma pneumoniae mixed infection can be judged.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer probe combination, which comprises the following components:
as set forth in SEQ ID NO: 1-2, a specific primer pair of mycoplasma pneumoniae;
as set forth in SEQ ID NO:3, a mycoplasma pneumoniae-sensitive detection probe;
as set forth in SEQ ID NO:4, detecting a drug-resistant mutation of a mycoplasma pneumoniae 2063 site;
and as set forth in SEQ ID NO:5, and detecting the drug-resistant mutation of the position 2064 of mycoplasma pneumoniae.
The primer and the probe of mycoplasma pneumoniae are used for qualitatively treating mycoplasma pneumoniae, and can detect whether the mycoplasma pneumoniae is drug-resistant mycoplasma pneumoniae or 23sRNA sensitive mycoplasma pneumoniae.
In the present invention, for different mycoplasma pneumoniae strains, SEQ ID NO: 1-2, wherein the amplified fragment of the primer pair is shown as SEQ ID NO:9 to 11.
As set forth in SEQ ID NO:9 is mycoplasma pneumoniae-sensitive;
as set forth in SEQ ID NO:10 is a mycoplasma pneumoniae 2063 mutant;
as set forth in SEQ ID NO:11 is a Mycoplasma pneumoniae 2064 mutant.
Preferably, the primer probe combination further includes:
as set forth in SEQ ID NO: 6-7, an internal standard amplification primer pair;
as set forth in SEQ ID NO: 8.
In the invention, the sequence of the internal standard is shown as SEQ ID NO. 12.
Preferably, the 3 'end of the probe is connected with a quenching group, and the 5' end is connected with a fluorescent group; wherein:
as set forth in SEQ ID NO:3, the 5' end of the probe is connected with a FAM fluorescent group;
as set forth in SEQ ID NO: 4-5, the 5' end of the probe is connected with a VIC fluorescent group;
as set forth in SEQ ID NO:8 is connected with a ROX fluorescent group at the 5' end of the probe.
The invention also provides application of the primer probe combination in preparation of a kit for detecting mycoplasma pneumoniae and drug-resistant mutation thereof.
The invention also provides a detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof, which comprises the primer probe combination and Real time PCR reaction reagent.
Preferably, the Real time PCR reaction reagent comprises one or more of the following reagents: dNTPs, UDG enzyme, taq enzyme, mgCl 2 。
Preferably, the kit further comprises a negative control and a positive control; the negative control is sterile water, the positive control is artificially synthesized concentration (0.1-10) x 10 6 Copies/mL pseudovirus.
In the specific examples provided herein, the positive control was a synthetic concentration of 1X 10 6 Copies/mL pseudovirus.
Preferably, the Real time PCR reaction system is:
dNTPs：200～300μM/mL；
UDG enzyme: 0.1-0.3U/. Mu.L;
taq enzyme: 15-20U/. Mu.L;
MgCl 2 ：0.1～3mmol/mL；
upstream and downstream primers: 350-400 nM/mL each;
and (3) probe: 150-200 nM/mL each;
sample to be measured: 20-30 mu L;
the water was made up to 40 μl.
Preferably, the Real time PCR reaction system is:
dNTPs：250μM/mL；
UDG enzyme: 0.2U/. Mu.L;
taq enzyme: 18.7U/. Mu.L;
MgCl 2 ：0.2mmol/mL；
upstream and downstream primers: 375nM/mL each;
and (3) probe: 187.5nM/mL each;
sample to be measured: 25 μL;
the water was made up to 40 μl.
Preferably, the Real time PCR reaction is performed as follows:
50℃2min；
95℃10min；
95℃15s,60℃15s,72℃10s (signal acquisition) for 40 cycles.
The invention also provides a method for detecting mycoplasma pneumoniae and drug-resistant mutation thereof, which comprises the following steps: detecting Real time PCR on the sample by adopting the primer probe combination, and judging whether the sample is infected by mycoplasma pneumoniae sensitive or drug-resistant according to the detected Ct value; and judging the proportion of the sensitive type and the drug resistant type in the mixed infection sample according to the difference (delta Ct) between the Ct value detected by the FAM channel and the Ct value detected by the VIC channel. The judging method comprises the following steps:
SEQ ID NO:3, but the CT value of the probe channel shown in SEQ ID NO: 4-5, and the CT value of the probe channel shown in SEQ ID NO:8, the CT value of the probe channel is less than or equal to 35, and the probe channel reports positive mycoplasma pneumoniae but not drug-resistant mycoplasma pneumoniae;
SEQ ID NO: 3> 36, but the probe channel shown in SEQ ID NO: 4-5, and the CT value of the probe channel shown in SEQ ID NO: the CT value of the probe channel shown in 8 is less than or equal to 35, and the probe channel reports that drug-resistant mycoplasma pneumoniae is positive, but the probe channel is not sensitive;
SEQ ID NO:3 and SEQ ID NO: the CT values of the probe channels shown in 4-5 are all less than or equal to 36, and SEQ ID NO: the CT value of the probe channel shown in 8 is less than or equal to 35, and the probe channel is reported to be positive for mycoplasma pneumoniae and is positive for the mixture of sensitive and drug-resistant; drug-resistant ratio in mixed positives = 2 ΔCt /1+2 ΔCt Sensitive ratio in mixed positives = 1/1+2 ΔCt
SEQ ID NO: 3. SEQ ID NO: the CT values of the probe channels shown in 4-5 are all more than or equal to 36, but SEQ ID NO:8, the CT value of the probe channel is less than or equal to 35, the concentration of the report sensitive mycoplasma pneumoniae sample and the drug-resistant mycoplasma pneumoniae sample are both lower than the detection lower limit, and the result is only used as a reference;
when SEQ ID NO:8, when the CT value of the probe channel is more than 36 and the negative control has the CT value or is in any one of a typical S amplification curve, a positive control has no CT value or no amplification curve, the detection result is invalid, the cause is searched and eliminated, and the test is repeated.
The invention provides a primer probe combination and a detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof. The primer probe combination comprises: as set forth in SEQ ID NO: 1-2, a specific primer pair of mycoplasma pneumoniae; as set forth in SEQ ID NO:3, a mycoplasma pneumoniae-sensitive detection probe; as set forth in SEQ ID NO:4, detecting a drug-resistant mutation of a mycoplasma pneumoniae 2063 site; and as set forth in SEQ ID NO:5, and detecting the drug-resistant mutation of the position 2064 of mycoplasma pneumoniae. The invention has the following beneficial effects:
the primer and the probe have good specificity, and can realize quick, accurate and sensitive identification and calculation of drug resistance proportion of mycoplasma pneumoniae and drug resistance mutation thereof by combining with a real time PCR detection method. Experiments show that the minimum detection limit of the primer probe for detecting mycoplasma pneumoniae sensitivity is 50copies/mL, and the minimum detection limit of mycoplasma pneumoniae drug-resistant type is 50copies/mL.
Detailed Description
The invention discloses a primer probe combination and a detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof, and a person skilled in the art can refer to the content of the primer probe combination and the detection kit and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The mycoplasma pneumoniae and the primer probe combination for the drug-resistant mutation thereof provided by the invention can be purchased from the market as test materials, instruments and the like used in the application of the detection kit.
The invention is further illustrated by the following examples:
example 1 preparation of Mycoplasma pneumoniae-sensitive and drug-resistant Mycoplasma pneumoniae-associated nucleic acid detection kit
Primer and probe sequences in the kit are shown in the following table 1:
TABLE 1 primer, probe sequences
| Name of the name | Numbering device | Nucleotide sequence |
| Upstream primer | SEQ ID NO：1 | AATCCAGGTACGGGTGAAGACA |
| Downstream primer | SEQ ID NO：2 | TGCTCCTACCTATTCTCTACATGATAATG |
| Sensitive probe | SEQ ID NO：3 | CAACGGGACGGAAA |
| 2063 mutant probe | SEQ ID NO：4 | CAACGGGACGGGAA |
| 2064 mutant probe | SEQ ID NO：5 | ACGGGACGGAGAG |
| Internal standard upstream primer | SEQ ID NO：6 | GAAGGCTCATGGCAAGAAAG |
| Internal standard downstream primer | SEQ ID NO：7 | CTCACTCAGTGTGGCAAAGG |
| Internal standard probe | SEQ ID NO：8 | CTTGAGGTTGTCCAGGTGAGCCAG |
The kit also comprises: 10mM dNTPs, 200U/. Mu.L UDG enzyme, 5U/. Mu.L Taq enzyme, 50mM MgCl 2 . The kit also comprises a negative control (sterile water) and a positive control (synthetic concentration 1×10) 6 Copies/mL of sensitive pseudovirus).
Example 2 detection method of the kit of the present invention
The detection method of the invention is Real time RT-PCR, and is specifically as follows:
1. the RT-PCR system is as follows:
dNTPs：250μM/mL；
UDG enzyme: 0.2U/. Mu.L;
taq enzyme: 18.7U/. Mu.L;
MgCl 2 ：0.2mmol/mL；
upstream and downstream primers: 375nM/mL each;
and (3) probe: 187.5nM/mL each;
sample to be measured: 25 μL;
the water was made up to 40 μl.
2. The Real Time RT-PCR reaction process is as follows:
50 ℃ for 2min;95 ℃ for 10min;95℃15s,60℃15s,72℃10s (signal acquisition) for 40 cycles.
3. Amplified fragment
SEQ ID NO:9 is mycoplasma pneumoniae-sensitive:
AATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGAAAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCA
SEQ ID NO:10 is a mycoplasma pneumoniae 2063 mutant:
AATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGAGAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCA
SEQ ID NO:11 is a mycoplasma pneumoniae 2064 mutant:
AATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGAAGGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCA
the sequence of the internal standard is shown as SEQ ID NO. 12:
GAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAG
4. fluorescence detection channel selection:
(1) Selecting FAM channel (reporTer: FAM, quenCher: none), and detecting sensitive mycoplasma pneumoniae;
(2) Selecting a VIC channel (reporTer: VIC, quenCher: none), and detecting drug-resistant mycoplasma pneumoniae;
(3) Selecting ROX channel, detecting internal standard;
(4) The reference fluorescence (PAssive ReferenCe) was set to none.
5. The specific test results were analyzed as follows:
SEQ ID NO:3, but the CT value of the probe channel shown in SEQ ID NO: 4-5, and the CT value of the probe channel shown in SEQ ID NO:8, the CT value of the probe channel is less than or equal to 35, and the probe channel reports positive mycoplasma pneumoniae but not drug-resistant mycoplasma pneumoniae;
SEQ ID NO: 3> 36, but the probe channel shown in SEQ ID NO: 4-5, and the CT value of the probe channel shown in SEQ ID NO: the CT value of the probe channel shown in 8 is less than or equal to 35, and the probe channel reports that drug-resistant mycoplasma pneumoniae is positive, but the probe channel is not sensitive;
SEQ ID NO:3 and SEQ ID NO: the CT values of the probe channels shown in 4-5 are all less than or equal to 36, and SEQ ID NO:8, a probe is led toThe CT value is less than or equal to 35, and the mycoplasma pneumoniae is reported to be positive and is a mixture positive of sensitive type and drug resistant type; drug-resistant ratio in mixed positives = 2 ΔCt /1+2 ΔCt Sensitive ratio in mixed positives = 1/1+2 ΔCt ；
SEQ ID NO: 3. SEQ ID NO: the CT values of the probe channels shown in 4-5 are all more than or equal to 36, but SEQ ID NO:8, the CT value of the probe channel is less than or equal to 35, the concentration of the report sensitive mycoplasma pneumoniae sample and the drug-resistant mycoplasma pneumoniae sample are both lower than the detection lower limit, and the result is only used as a reference;
when SEQ ID NO:8, when the CT value of the probe channel is more than 36 and the negative control has the CT value or is in any one of a typical S amplification curve, a positive control has no CT value or no amplification curve, the detection result is invalid, the cause is searched and eliminated, and the test is repeated.
Example 3 feasibility test of the kit of the invention
1. Minimum limit of detection (LOD) test
(1) Preparing a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent: a Mycoplasma pneumoniae-drug-resistant Mycoplasma pneumoniae duplex detection was prepared by the method of example 1.
(2) Bacterial sample extraction
Mixing 5 sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae human throat swab eluents in the tube uniformly by using a pipettor, taking out 100 mu L of the mixture in a new centrifuge tube, centrifuging at 12000rpm for 5min, and carefully discarding the supernatant; 200. Mu.L of bacterial lysate (from Beijing Bai Albo technology Co., ltd.) was added to the pellet, thoroughly mixed, and centrifuged at 10000rpm in a water bath at 100℃for 5min for further use.
(3) Sample detection
25. Mu.L of the treated specimen supernatant was added to a reaction tube of a duplex nucleic acid detection reagent for mycoplasma pneumoniae and mycoplasma pneumoniae of drug-resistant type, each concentration was 20 multiplex wells, and 25. Mu.L of purified water was added to the detection solution as a negative control, and detection was performed according to the detection method in example 2.
(4) Analysis of results
The detection of the samples of each concentration gradient of the mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent by using the kit prepared in example 1 and the detection method in example 2 shows that the detection sensitivity (LOD) mycoplasma pneumoniae of the detection method is 50copies/mL, and the drug-resistant mycoplasma pneumoniae is 50copies/mL, and the specific data are shown in tables 2 and 3.
Table 2: mycoplasma pneumoniae detection limit confirmation
| Sample concentration (copies/mL) | Detecting the repetition number | Number of positive tests | Positive detection rate |
| 20 | 21 | 0 | 0.00％ |
| 30 | 21 | 15 | 71.43％ |
| 50 | 21 | 21 | 100.00％ |
| 100 | 21 | 21 | 100.00％ |
| 200 | 21 | 21 | 100.00％ |
TABLE 3 confirmation of detection limits of Mycoplasma pneumoniae resistant to human
| Sample concentration (copies/mL) | Detecting the repetition number | Number of positive tests | Positive detection rate |
| 20 | 21 | 0 | 0.00％ |
| 30 | 21 | 18 | 85.71％ |
| 50 | 21 | 21 | 100.00％ |
| 100 | 21 | 21 | 100.00％ |
| 200 | 21 | 21 | 100.00％ |
2. Cross-reaction with other diseases
(1) The Mycoplasma pneumoniae and drug-resistant Mycoplasma pneumoniae duplex detection reagent was prepared by the method of example 1.
(2) Cross pathogen sample extraction
Mixing sensitive mycoplasma pneumoniae, drug-resistant mycoplasma pneumoniae, staphylococcus aureus, coagulase-negative staphylococcus (CNS), drug-resistant staphylococcus epidermidis (MRSE), diphtheria bacillus, haemophilus influenzae, bacillus proteus, streptococcus pneumoniae, escherichia coli, pseudomonas aeruginosa and candida albicans throat swab eluate in a tube uniformly by using a pipette, taking out 100 mu L of the eluate in a new centrifuge tube, centrifuging at 12000rpm for 5min, and carefully discarding the supernatant; 200 mu L of virus lysate is added into the precipitate, the mixture is fully and uniformly mixed, water bath is carried out for 5 minutes at 100 ℃, and centrifugation is carried out for 5 minutes at 10000rpm for standby.
(3) Sample detection
25 mu L of the treated specimen supernatant is added into a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reaction tube, 25 mu L of purified water is added into detection liquid to serve as negative control, extracted sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae serve as positive control tests, and detection is carried out according to the detection method in example 2.
(4) Analysis of results
The detection of pathogens other than mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae by using the kit prepared in example 1 and the detection method in example 2 shows that: the kit provided by the invention has no cross reaction on pathogen infection samples such as sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae positive control, negative control is negative, staphylococcus aureus, coagulase Negative Staphylococcus (CNS), drug-resistant staphylococcus epidermidis (MRSE), diphtheria bacillus, haemophilus influenzae, denatured bacillus, streptococcus pneumoniae, escherichia coli, pseudomonas aeruginosa and the like, and shows that the kit has high specificity, and specific results are shown in Table 4.
Table 4: cross reaction experiment
3. Tamper resistance to potentially foreign substances
(1) Preparing a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent: the preparation of the reagent for detecting the duplex nucleic acid of the mycoplasma pneumoniae and the drug-resistant mycoplasma pneumoniae is carried out by adopting the method of the example 1.
(2) Sample processing
And selecting two concentration values of high and low values of the sensitive mycoplasma pneumoniae and the drug-resistant mycoplasma pneumoniae. The two concentration values of the sensitive mycoplasma pneumoniae are respectively 1 multiplied by 10 6 The concentration values of the two drug-resistant mycoplasma pneumoniae are respectively 1 multiplied by 10, wherein the concentration values of the two drugs are respectively 1 multiplied by 10 6 The copies/mL and 50copies/mL. At the same time, interfering substances were added to the corresponding virus samples at a peak concentration (Cmax) of 3 times, and the samples were treated by the cross-reaction method of example 3 and tested according to the test method of example 2.
(3) Analysis of results
Interference judgment: the interference rate% is smaller than the accuracy bias allowed by the line standard of the project (set as 10%), and the pass can be judged.
The interference rate calculation formula: (interference sample concentration-control sample concentration)/control sample concentration x 100%.
Experiments show that when samples contain common antiviral drugs such as ribavirin, menthol, tenofovir, lamivudine, benzocaine, adefovir Wei Cu, dexamethasone, entecavir, epinephrine, zanamivir (200 ug/mL) and the like, the detection sensitivity of the kit is not obviously interfered, and the detection sensitivity is specifically shown in table 5.
Table 5: anti-interference experiment of exogenous substances
| Drug name | Interference ratio (%) | Drug name | Interference ratio (%) |
| Ribavirin | 2.2 | Adeford Wei Cu | 2.4 |
| Menthol crystal | 1.6 | Dexamethasone | 2.2 |
| Tenofovir | 1.8 | Entecavir | 1.1 |
| Lamivudine | 1.9 | Epinephrine system | 1.5 |
| Benzocaine | 2.3 | Zanamivir | 1.4 |
4. Interference resistance against potentially endogenous substances
(1) Preparing a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent: a Mycoplasma pneumoniae-drug-resistant Mycoplasma pneumoniae duplex detection reagent was prepared by the method of example 1.
(2) Sample processing
And selecting two concentration values of high and low values of mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae. Mycoplasma pneumoniae concentration values were 1X 10 respectively 8 The concentration values of the two drug-resistant mycoplasma pneumoniae are respectively 1 multiplied by 10, wherein the concentration values of the two drugs are respectively 1 multiplied by 10 8 The copies/mL and 10copies/mL. At the same time, interfering substances were added to the corresponding virus samples at a peak concentration (Cmax) of 3 times, and the samples were treated by the cross-reaction method of example 3 and tested according to the test method of example 2. Meanwhile, 200mg/dL hemoglobin, 3000mg/dL triglyceride, 20mg/dL bilirubin and other interfering substances are added into corresponding virus samples, and the samples are processed by the cross reaction method in the example 3 and detected according to the detection method in the example 2.
(3) Analysis of results
Interference judgment: the interference rate% is smaller than the accuracy bias allowed by the line standard of the project (set as 10%), and the pass can be judged.
The interference rate calculation formula: (interference sample concentration-control sample concentration)/control sample concentration x 100%.
Experiments show that when the sample contains 200mg/dL hemoglobin, 3000mg/dL triglyceride, 20mg/dL bilirubin and other endogenous interfering substances, the detection sensitivity of the kit of the invention is not obviously interfered, and the specific is shown in Table 6.
Table 6: anti-interference experiment of endogenous substances
| Interfering substances | Interference ratio (%) |
| Hemoglobin (hemoglobin) | 2.8 |
| Triglycerides (Triglycerides) | 2.1 |
| Bilirubin | 3.5 |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Zhengzhou Anji bioengineering Co., ltd
<120> primer probe combination and detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof
<130> MP2020848
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
aatccaggta cgggtgaaga ca 22
<210> 2
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tgctcctacc tattctctac atgataatg 29
<210> 3
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
caacgggacg gaaa 14
<210> 4
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
caacgggacg ggaa 14
<210> 5
<211> 13
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
acgggacgga gag 13
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gaaggctcat ggcaagaaag 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ctcactcagt gtggcaaagg 20
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
cttgaggttg tccaggtgag ccag 24
<210> 9
<211> 116
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
aatccaggta cgggtgaaga cacccgttag gcgcaacggg acggaaagac cccgtgaagc 60
tttactgtag cttaatattg atcaggacat tatcatgtag agaataggta ggagca 116
<210> 10
<211> 116
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
aatccaggta cgggtgaaga cacccgttag gcgcaacggg acggagagac cccgtgaagc 60
tttactgtag cttaatattg atcaggacat tatcatgtag agaataggta ggagca 116
<210> 11
<211> 116
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
aatccaggta cgggtgaaga cacccgttag gcgcaacggg acggaaggac cccgtgaagc 60
tttactgtag cttaatattg atcaggacat tatcatgtag agaataggta ggagca 116
<210> 12
<211> 91
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gaaggctcat ggcaagaaag tgctcggtgc ctttagtgat ggcctggctc acctggacaa 60
cctcaagggc acctttgcca cactgagtga g 91
Claims (6)
1. A primer probe combination, comprising:
as set forth in SEQ ID NO: 1-2, a specific primer pair of mycoplasma pneumoniae;
as set forth in SEQ ID NO:3, a mycoplasma pneumoniae-sensitive detection probe;
as set forth in SEQ ID NO:4, detecting a drug-resistant mutation of a mycoplasma pneumoniae 2063 site;
and as set forth in SEQ ID NO:5, detecting a drug-resistant mutation of a mycoplasma pneumoniae 2064 site;
as set forth in SEQ ID NO: 6-7, an internal standard amplification primer pair;
as set forth in SEQ ID NO:8, an internal standard probe shown in the figure;
the 3 'end of the probe is connected with a quenching group, and the 5' end of the probe is connected with a fluorescent group; wherein:
as set forth in SEQ ID NO:3, the 5' end of the probe is connected with a FAM fluorescent group;
as set forth in SEQ ID NO: 4-5, connecting a VIC fluorescent group at the 5' end of the probe;
as set forth in SEQ ID NO:8 is connected with a ROX fluorescent group at the 5' end of the probe.
2. The primer probe combination of claim 1, wherein the sequence of SEQ ID NO: 1-2, wherein the amplified fragment of the primer pair is shown as SEQ ID NO: 9-11.
3. Use of the primer probe combination according to claim 1 or 2 for preparing a kit for detecting mycoplasma pneumoniae and drug-resistant mutations thereof.
4. A kit for detecting mycoplasma pneumoniae and drug-resistant mutations thereof, which is characterized by comprising the primer probe combination of claim 1 or 2 and Real time PCR reaction reagent;
the Real time PCR reaction system is as follows:
dNTPs：200~300μM/mL；
UDG enzyme: 0.1 to 0.3U/. Mu.L;
taq enzyme: 15-20U/. Mu.L;
MgCl 2 ：0.1~3mmol/μL；
upstream and downstream primers: 350-400 nM/mL each;
and (3) probe: 150-200 nM/mL each;
sample to be measured: 20-30 mu L;
water make up to 40 μl;
judging the proportion of sensitive type and drug resistant type in the mixed infection sample by subtracting the Ct value difference (delta Ct) detected by the VIC channel from the Ct value detected by the FAM channel: drug-resistant ratio in mixed positives = 2 ΔCt /1+2 ΔCt Sensitive ratio in mixed positives = 1/1+2 ΔCt 。
5. The test kit of claim 4, wherein the kit further comprises a negative control and a positive control; the negative control is sterile water, and the positive control is artificially synthesized concentration (0.1-10) multiplied by 10 6 Copies/mL pseudovirus.
6. The test kit of claim 4 or 5, wherein the Real time PCR reaction is programmed to:
50℃ 2min；
95℃ 10min；
95℃15s,60℃15s,72℃10s, 40 cycles total.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002522A (en) * | 2009-11-10 | 2011-04-06 | 复旦大学附属华山医院 | Method for detecting resistant mutant of mycoplasma pneumoniae |
| CN106191239A (en) * | 2016-07-11 | 2016-12-07 | 宁波基内生物技术有限公司 | A kind of real-time PCR detection mycoplasma pneumoniae nucleic acid and the primer of medicament-resistant mutation, probe, method and test kit |
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| EP1246935B1 (en) * | 1999-09-28 | 2013-08-14 | Geneohm Sciences Canada Inc. | Highly conserved genes and their use to generate probes and primers for detection of microorganisms |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002522A (en) * | 2009-11-10 | 2011-04-06 | 复旦大学附属华山医院 | Method for detecting resistant mutant of mycoplasma pneumoniae |
| CN106191239A (en) * | 2016-07-11 | 2016-12-07 | 宁波基内生物技术有限公司 | A kind of real-time PCR detection mycoplasma pneumoniae nucleic acid and the primer of medicament-resistant mutation, probe, method and test kit |
Non-Patent Citations (3)
| Title |
|---|
| Yu Suzuki等.Development of an endpoint genotyping assay to detect the Mycoplasma pneumoniae 23S rRNA gene and distinguish the existence of macrolide resistance-associated mutations at position 2063.《Journal of Microbiological Methods》.2016,第130-134页. * |
| 刘杨.肺炎支原体耐药性及耐药机制研究.《中国博士学位论文全文数据库医药卫生科技辑》.2010,第56-62页. * |
| 李静宜等.实时定量PCＲ 检测肺炎支原体2063 位点点突变的方法学研究.《中国全科医学》.2013,第16卷(第11期),第3774-3777页. * |
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