CN112458194A - Mycoplasma pneumoniae and drug-resistant mutation primer-probe combination thereof and detection kit - Google Patents
Mycoplasma pneumoniae and drug-resistant mutation primer-probe combination thereof and detection kit 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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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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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 shown in SEQ ID NO: 1-2, and a specific primer pair of Mycoplasma pneumoniae; as shown in SEQ ID NO: 3, and a mycoplasma pneumoniae sensitive detection probe; as shown in SEQ ID NO: 4, a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2063 site; and as shown in SEQ ID NO: 5, and a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2064 site. The primer and the probe have good specificity, and can realize quick, accurate and sensitive identification of the mycoplasma pneumoniae and the drug-resistant mutation thereof and calculation of the drug-resistant ratio by combining 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 have developed resistance to tetracycline antibiotics, and the phenomenon of enhanced resistance has attracted more and more attention.
The detection method of the mycoplasma pneumoniae is various, the isolation culture is the 'gold standard' for microbial detection, although the method is reliable, the method is time-consuming and labor-consuming and cannot meet the requirement of rapid detection, 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 positive sometimes occurs. In order to overcome the problems, the multiplex fluorescence quantitative RT-PCR technology is used for diagnosing infection of mycoplasma pneumoniae and drug resistance thereof, the method has the characteristics of rapidness, sensitivity, strong specificity and the like, the probe can distinguish the sensitive type and the drug resistance type and can calculate the proportion of drug-resistant bacteria, and the detection efficiency is greatly improved.
However, at present, unreasonable design of primer probes for identifying mycoplasma pneumoniae and drug resistance thereof causes that most detection reagents cannot realize accurate, rapid and sensitive detection. The detection sensitivity of the primer probe disclosed by the invention with the authorization publication number CN 102002522B is 100 Copies/ml. The primer probe disclosed by the publication number CN 106191239A has the detection sensitivity of 1000Copies/ml, needs to be respectively detected by 3 tubes in the detection process, and has large workload and low detection flux. The primer probe disclosed by the publication No. CN 103820557A can only detect sensitive strains and 2063 site mutant strains. And the three patent methods can not judge the drug resistance ratio of the mycoplasma pneumoniae mixed infection.
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 mutations thereof. The minimum detection limit of the primer probe combination on the sensitive type and the mutant type of the mycoplasma pneumoniae is 50copies/mL, and the drug resistance ratio of the 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:
as shown in SEQ ID NO: 1-2, and a specific primer pair of Mycoplasma pneumoniae;
as shown in SEQ ID NO: 3, and a mycoplasma pneumoniae sensitive detection probe;
as shown in SEQ ID NO: 4, a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2063 site;
and as shown in SEQ ID NO: 5, and a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2064 site.
The primer and the probe of the mycoplasma pneumoniae are used for characterizing the mycoplasma pneumoniae, and can be used for detecting both drug-resistant mycoplasma pneumoniae and 23 sRNA-sensitive mycoplasma pneumoniae.
In the present invention, the sequences of SEQ ID NOs: 1-2 is shown as SEQ ID NO: 9 to 11.
As shown in SEQ ID NO: 9, the amplified fragment is sensitive to mycoplasma pneumoniae;
as shown in SEQ ID NO: 10 is a mutant mycoplasma pneumoniae 2063;
as shown in SEQ ID NO: 11 is a mutant mycoplasma pneumoniae 2064.
Preferably, the primer probe combination further comprises:
as shown in SEQ ID NO: 6-7 of an internal standard amplification primer pair;
as shown in SEQ ID NO: 8, and (b) an internal standard probe.
In the invention, the sequence of the internal standard is shown in SEQ ID NO. 12.
Preferably, 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 shown in SEQ ID NO: 3, connecting the 5' end of the probe with FAM fluorescent group;
as shown in SEQ ID NO: the 5' end of the probe shown in 4-5 is connected with a VIC fluorescent group;
as shown in SEQ ID NO: the 5' end of the probe shown in 8 is connected with a ROX fluorescent group.
The invention also provides application of the primer probe combination in preparing a kit for detecting mycoplasma pneumoniae and drug-resistant mutation thereof.
The invention also provides a detection kit for the mycoplasma pneumoniae and the drug resistance mutation thereof, and the kit comprises the primer probe combination and a 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, MgCl2。
Preferably, 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) x 106Copies/mL of pseudovirus.
In the specific embodiment provided by the present invention, the positive control is an artificially synthesized concentration of 1X 106Copies/mL of pseudovirus.
Preferably, the Real time PCR reaction system is as follows:
dNTPs：200～300μM/mL；
UDG enzyme: 0.1-0.3U/muL;
taq enzyme: 15-20U/muL;
MgCl2：0.1～3mmol/mL；
upstream and downstream primers: each 350-400 nM/mL;
and (3) probe: each 150-200 nM/mL;
a sample to be detected: 20-30 mu L;
make up to 40. mu.L of water.
Preferably, the system of Real time PCR reaction is:
dNTPs：250μM/mL；
UDG enzyme: 0.2U/. mu.L;
taq enzyme: 18.7U/. mu.L;
MgCl2：0.2mmol/mL；
upstream and downstream primers: 375nM/mL each;
and (3) probe: 187.5nM/mL each;
a sample to be detected: 25 mu L of the solution;
make up to 40. mu.L of water.
Preferably, the procedure for the Real time PCR reaction is:
50℃2min；
95℃10min；
95 ℃ 15s, 60 ℃ 15s, 72 ℃ 10s (signal acquisition), for a total of 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 type or drug resistant type according to the detected Ct value; and (3) 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: CT value of the probe channel shown in 3 is less than or equal to 36, but the nucleotide sequence shown in SEQ ID NO: the CT value of the probe channel shown in 4-5 is more than 36, and the nucleotide sequence shown in SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, and the probe channel is reported to be positive by mycoplasma pneumoniae but not drug-resistant mycoplasma pneumoniae;
SEQ ID NO: CT values for probe channels indicated by 3 were > 36, but the sequences shown in SEQ ID NO: the CT value of the probe channel shown by 4-5 is less than or equal to 36, and the molecular weight of SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, and the result is reported to be positive for drug-resistant mycoplasma pneumoniae but non-sensitive mycoplasma pneumoniae;
SEQ ID NO: 3 and SEQ ID NO: the CT values of the probe channels shown by 4-5 are all less than or equal to 36, and the CT values of SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, and the probe channel is reported to be positive by mycoplasma pneumoniae and is mixed positive by sensitive type and drug-resistant type; the proportion of the drug resistant type in the mixed positive traditional Chinese medicine is 2ΔCt/1+2ΔCtThe proportion of the mixed positive medium sensitive type is 1/1+2ΔCt
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 the CT values of the probe channels shown in SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, the concentrations of the reported sensitive mycoplasma pneumoniae sample and the reported drug-resistant mycoplasma pneumoniae sample are both lower than the lower detection limit, and the results are only used as reference;
when SEQ ID NO: and the CT value of the probe channel shown by 8 is more than 36, when the negative control has the CT value or presents a typical S amplification curve, and the positive control has no CT value or no amplification curve, the detection result is invalid, the reason 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 shown in SEQ ID NO: 1-2, and a specific primer pair of Mycoplasma pneumoniae; as shown in SEQ ID NO: 3, and a mycoplasma pneumoniae sensitive detection probe; as shown in SEQ ID NO: 4, a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2063 site; and as shown in SEQ ID NO: 5, and a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2064 site. The invention has the following beneficial effects:
the primer and the probe have good specificity, and can realize quick, accurate and sensitive identification of the mycoplasma pneumoniae and the drug-resistant mutation thereof and calculation of the drug-resistant ratio by combining a real time PCR detection method. Experiments show that the minimum detection limit of the primer probe for detecting the mycoplasma pneumoniae sensitive type is 50copies/mL, and the minimum detection limit of the mycoplasma pneumoniae drug resistant type is 50 copies/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 realize the primer probe combination and the detection kit by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The primer probe combination for mycoplasma pneumoniae and drug-resistant mutation thereof and the detection kit provided by the invention can be used for detecting the drug-resistant mutation of mycoplasma pneumoniae.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of Duplex nucleic acid detection kit for sensitive Mycoplasma pneumoniae and drug-resistant Mycoplasma pneumoniae
The sequences of the primers and the probes in the kit are shown in the following table 1:
TABLE 1 primer, Probe sequences
| Name (R) | Numbering | 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 MgCl2. The kit also includes a negative control (sterile water) and a positive control (artificially synthesized at a concentration of 1X 10)6Sensitive pseudovirus of Copies/mL).
Example 2 detection method of the kit of the present invention
The detection method is Real time RT-PCR, and specifically comprises the following steps:
1. the RT-PCR system is as follows:
dNTPs：250μM/mL；
UDG enzyme: 0.2U/. mu.L;
taq enzyme: 18.7U/. mu.L;
MgCl2：0.2mmol/mL；
upstream and downstream primers: 375nM/mL each;
and (3) probe: 187.5nM/mL each;
a sample to be detected: 25 mu L of the solution;
make up to 40. mu.L of water.
2. The Real Time RT-PCR reaction process is as follows:
2min at 50 ℃; 10min at 95 ℃; 95 ℃ 15s, 60 ℃ 15s, 72 ℃ 10s (signal acquisition), for a total of 40 cycles.
3. Amplification of fragments
SEQ ID NO: the amplified fragment shown in 9 is mycoplasma pneumoniae sensitive:
AATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGAAAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCA
SEQ ID NO: 10 is a mutant mycoplasma pneumoniae 2063:
AATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGAGAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCA
SEQ ID NO: 11 is a mutant mycoplasma pneumoniae 2064:
AATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGAAGGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCA
the sequence of the internal standard is shown in SEQ ID NO: 12:
GAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAG
4. fluorescence detection channel selection:
(1) selecting a FAM channel (ReporTer: FAM, Quencher: none), and detecting sensitive mycoplasma pneumoniae;
(2) selecting VIC channel (ReporTer: VIC, Quencher: none), and detecting drug-resistant mycoplasma pneumoniae;
(3) selecting a ROX channel, and detecting an internal standard;
(4) the ReferenCe fluorescence (PAStive ReferenCe) was set to none.
5. The specific test results were analyzed as follows:
SEQ ID NO: CT value of the probe channel shown in 3 is less than or equal to 36, but the nucleotide sequence shown in SEQ ID NO: the CT value of the probe channel shown in 4-5 is more than 36, and the nucleotide sequence shown in SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, and the probe channel is reported to be positive by mycoplasma pneumoniae but not drug-resistant mycoplasma pneumoniae;
SEQ ID NO: CT values for probe channels indicated by 3 were > 36, but the sequences shown in SEQ ID NO: the CT value of the probe channel shown by 4-5 is less than or equal to 36, and the molecular weight of SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, and the result is reported to be positive for drug-resistant mycoplasma pneumoniae but non-sensitive mycoplasma pneumoniae;
SEQ ID NO: 3 and SEQ ID NO: the CT values of the probe channels shown by 4-5 are all less than or equal to 36, and the CT values of SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, and the probe channel is reported to be positive by mycoplasma pneumoniae and is mixed positive by sensitive type and drug-resistant type; the proportion of the drug resistant type in the mixed positive traditional Chinese medicine is 2ΔCt/1+2ΔCtThe proportion of the mixed positive medium sensitive type is 1/1+2ΔCt；
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 the CT values of the probe channels shown in SEQ ID NO: the CT value of the probe channel shown by 8 is less than or equal to 35, the concentrations of the reported sensitive mycoplasma pneumoniae sample and the reported drug-resistant mycoplasma pneumoniae sample are both lower than the lower detection limit, and the results are only used as reference;
when SEQ ID NO: and the CT value of the probe channel shown by 8 is more than 36, when the negative control has the CT value or presents a typical S amplification curve, and the positive control has no CT value or no amplification curve, the detection result is invalid, the reason is searched and eliminated, and the test is repeated.
EXAMPLE 3 feasibility test of the kit of the invention
1. Limit of detection (LOD) test
(1) Preparing a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent: a nucleic acid test for both sensitive and drug-resistant Mycoplasma pneumoniae was prepared by the method of example 1.
(2) Bacterial sample extraction
Mixing 5 sensitive Mycoplasma pneumoniae and drug-resistant Mycoplasma pneumoniae human pharynx swab eluents with different concentrations in a tube uniformly by using a pipettor, taking out 100 microliter of the mixture in a new centrifuge tube, centrifuging at 12000rpm for 5min, and carefully discarding the supernatant; adding 200 μ L bacterial lysate (from Beijing Baiolaibobo science and technology Co., Ltd.), mixing well, water bathing at 100 deg.C for 5min, and centrifuging at 10000rpm for 5min for use.
(3) Sample detection
mu.L of the treated specimen supernatant was added to a nucleic acid detecting reagent reaction tube for both sensitive and drug-resistant Mycoplasma pneumoniae in 20-well-per-concentration, and 25. mu.L of purified water was added to the test solution as a negative control to carry out the test in accordance with the test method described in example 2.
(4) Analysis of results
The detection of samples with each concentration gradient of the nucleic acid detection reagent of the combination of sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae by using the kit prepared in example 1 and the detection method of example 2 shows that the detection sensitivity (LOD) of the detection method is 50copies/mL for mycoplasma pneumoniae and 50copies/mL for drug-resistant mycoplasma pneumoniae, and the specific data are shown in tables 2 and 3.
Table 2: confirmation of detection limit of mycoplasma pneumoniae
| Sample concentration (copies/mL) | Detecting the number of repetitions | Number of positive tests | Rate of positive detection |
| 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 human drug-resistant Mycoplasma pneumoniae
| Sample concentration (copies/mL) | Detecting the number of repetitions | Number of positive tests | Rate of positive detection |
| 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-reactive conditions with other diseases
(1) The sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent is prepared by the method of example 1.
(2) Cross pathogen sample extraction
Uniformly mixing sensitive mycoplasma pneumoniae, drug-resistant mycoplasma pneumoniae, staphylococcus aureus, Coagulase Negative Staphylococcus (CNS), drug-resistant staphylococcus epidermidis (MRSE), diphtheria bacillus, haemophilus influenzae, denaturated bacillus, streptococcus pneumoniae, escherichia coli, pseudomonas aeruginosa and candida albicans throat swab eluates in a pipette, taking out 100 mu L of the eluent to a new centrifugal tube, centrifuging at 12000rpm for 5min, and carefully discarding the supernatant; adding 200 μ L virus lysate into the precipitate, mixing well, water bathing at 100 deg.C for 5min, and centrifuging at 10000rpm for 5min for use.
(3) Sample detection
25 μ L of the treated specimen supernatant was added to a nucleic acid detection reaction tube for detecting both sensitive and drug-resistant Mycoplasma pneumoniae, 25 μ L of purified water was added to the test solution as a negative control, and the extracted sensitive and drug-resistant Mycoplasma pneumoniae were used as a positive control for the test, and the test was carried out according to the test method described in example 2.
(4) Analysis of results
By using the kit prepared in example 1 and the detection method of example 2 to detect other pathogens other than mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae, the results show that: the kit disclosed by the invention is positive for positive controls of sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae, negative controls are negative, and staphylococcus aureus, coagulase-negative staphylococcus (CNS), drug-resistant staphylococcus epidermidis (MRSE), diphtheria bacillus, haemophilus influenzae, denaturizing bacillus, streptococcus pneumoniae, escherichia coli, pseudomonas aeruginosa and other pathogen infected samples have no cross reaction, so that the kit has high specificity, and specific results are shown in table 4.
Table 4: cross reaction experiment
3. Interference immunity to potentially foreign substances
(1) Preparing a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent: the reagent is prepared by preparing a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent by the method of example 1.
(2) Sample processing
Selecting two concentration values of high value and low value of sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae. The two concentration values of sensitive mycoplasma pneumoniae are respectively 1 × 106Two concentration values of copies/mL and 50copies/mL, and drug-resistant Mycoplasma pneumoniae are respectively 1 × 106copies/mL and 50 copies/mL. At the same time willThe interfering substance was added to the corresponding virus sample at a peak concentration (Cmax) of 3 times, and the sample was treated by the cross-reaction method in example 3 and detected by the detection method in example 2.
(3) Analysis of results
And (3) interference judgment: the percentage of interference is less than the accuracy bias (set to 10%) allowed by the column index of the item, and it can be determined as passing.
The interference rate calculation formula is as follows: (concentration of interfering sample-concentration of control sample)/concentration of control sample x 100%.
Tests show that when a sample contains common antiviral drugs such as ribavirin, menthol, tenofovir, lamivudine, benzocaine, adefovir vinegar, dexamethasone, entecavir, epinephrine, zanamivir (200ug/mL), and the like, the detection sensitivity of the kit provided by the invention is not obviously interfered, and the details are shown in Table 5.
Table 5: anti-interference experiment of exogenous substance
| Name of drug | Interference ratio (%) | Name of drug | Interference ratio (%) |
| Ribavirin | 2.2 | Adefovir vinegar | 2.4 |
| Menthol crystal | 1.6 | Dexamethasone | 2.2 |
| Tenofovir | 1.8 | Entecavir | 1.1 |
| Lamivudine | 1.9 | Adrenalin | 1.5 |
| Benzocaine | 2.3 | Zanamivir | 1.4 |
4. Interference resistance to potential endogenous substances
(1) Preparing a sensitive mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae duplex nucleic acid detection reagent: a nucleic acid detecting reagent for detecting both sensitive Mycoplasma pneumoniae and drug-resistant Mycoplasma pneumoniae was prepared by the method of example 1.
(2) Sample processing
Selecting two concentration values of high value and low value of mycoplasma pneumoniae and drug-resistant mycoplasma pneumoniae. The two concentration values of Mycoplasma pneumoniae are 1 × 10 respectively8Two concentration values of copies/mL and 50copies/mL, and drug-resistant Mycoplasma pneumoniae are respectively 1 × 108copies/mL and 10 copies/mL. At the same time, the interfering substance was added to the corresponding virus sample at a peak concentration (Cmax) of 3 times, and the sample was treated by the cross-reaction method in example 3 and detected by the detection method in example 2. Simultaneously adding interfering substances such as 200mg/dL hemoglobin, 3000mg/dL triglyceride, 20mg/dL bilirubin and the like into corresponding virus samples, processing the samples by adopting the cross reaction method in the example 3, and detecting according to the detection method in the example 2The method is used for detection.
(3) Analysis of results
And (3) interference judgment: the percentage of interference is less than the accuracy bias (set to 10%) allowed by the column index of the item, and it can be determined as passing.
The interference rate calculation formula is as follows: (concentration of interfering sample-concentration of control sample)/concentration of control sample x 100%.
Experiments show that when a sample contains endogenous interference substances such as 200mg/dL hemoglobin, 3000mg/dL triglyceride, 20mg/dL bilirubin and the like, the detection sensitivity of the kit provided by the invention is not obviously interfered, and the details are shown in Table 6.
Table 6: anti-interference experiments with endogenous substances
| Interfering substances | Interference ratio (%) |
| Hemoglobin | 2.8 |
| Triglycerides | 2.1 |
| Bilirubin | 3.5 |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Zhengzhou Antu bioengineering GmbH
<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 (10)
1. A primer probe combination, comprising:
as shown in SEQ ID NO: 1-2, and a specific primer pair of Mycoplasma pneumoniae;
as shown in SEQ ID NO: 3, and a mycoplasma pneumoniae sensitive detection probe;
as shown in SEQ ID NO: 4, a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2063 site;
and as shown in SEQ ID NO: 5, and a detection probe of the drug-resistant mutation of the mycoplasma pneumoniae 2064 site.
2. The primer probe combination of claim 1, wherein the primer sequence of SEQ ID NO: 1-2 is shown as SEQ ID NO: 9 to 11.
3. The primer-probe combination of claim 1 or 2, wherein the primer-probe combination further comprises:
as shown in SEQ ID NO: 6-7 of an internal standard amplification primer pair;
as shown in SEQ ID NO: 8, and (b) an internal standard probe.
4. The primer probe combination of any one of claims 1 to 3, wherein the probe is linked to a quencher group at the 3 'end and a fluorophore group at the 5' end; wherein:
as shown in SEQ ID NO: 3, connecting the 5' end of the probe with FAM fluorescent group;
as shown in SEQ ID NO: the 5' end of the probe shown in 4-5 is connected with a VIC fluorescent group;
as shown in SEQ ID NO: the 5' end of the probe shown in 8 is connected with a ROX fluorescent group.
5. Use of the primer probe combination of any one of claims 1 to 4 for preparing a kit for detecting mycoplasma pneumoniae and drug-resistant mutations thereof.
6. A detection kit for Mycoplasma pneumoniae and drug-resistant mutations thereof, which is characterized by comprising the primer-probe combination as claimed in any one of claims 1 to 4 and Real time PCR reaction reagents.
7. The detection kit of claim 6, wherein the Real time PCR reaction reagent comprises one or more of the following reagents: dNTPs, UDG enzyme, Taq enzyme, MgCl2。
8. The test kit of claim 6, 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) x 106Copies/mL of pseudovirus.
9. The detection kit according to any one of claims 6 to 8, wherein the Real time PCR reaction system is as follows:
dNTPs：200～300μM/mL；
UDG enzyme: 0.1-0.3U/muL;
taq enzyme: 15-20U/muL;
MgCl2：0.1～3mmol/μL；
upstream and downstream primers: each 350-400 nM/mL;
and (3) probe: each 150-200 nM/mL;
a sample to be detected: 20-30 mu L;
make up to 40. mu.L of water.
10. The detection kit of claim 9, wherein the Real time PCR reaction is programmed to:
50℃2min；
95℃10min；
95 ℃ for 15s, 60 ℃ for 15s, 72 ℃ for 10s, for a total of 40 cycles.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090068641A1 (en) * | 1999-09-28 | 2009-03-12 | Bergeron Michel G | Highly conserved genes and their use to generate probes and primers for detection of microorganisms |
| 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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2020
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090068641A1 (en) * | 1999-09-28 | 2009-03-12 | Bergeron Michel G | Highly conserved genes and their use to generate probes and primers for detection of microorganisms |
| 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" * |
| 刘杨: "肺炎支原体耐药性及耐药机制研究" * |
| 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" * |
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