Specific probe, primer probe combination for detecting mycoplasma pneumoniae drug-resistant mutation and bordetella pertussis drug-resistant mutation and kit

Abstract

The invention relates to the technical field of molecular biology, in particular to a specific probe, a primer probe combination for detecting mycoplasma pneumoniae drug-resistant mutation and bordetella pertussis drug-resistant mutation and a kit. The primer probe combination and the kit provided by the invention can distinguish mutation at sites 2063 and 2064 of mycoplasma pneumoniae non-mutation sensitive strains and 23s rRNA genes as well as mutation at sites 2047 of bordetella pertussis non-mutation sensitive strains and 23s rRNA genes, and are relatively high in detection sensitivity; the sensitivity of a mycoplasma pneumoniae sensitive strain, a drug-resistant mutant strain and a bordetella pertussis sensitive strain can reach 25 copies / mL, high sensitivity and specificity are achieved by using locked nucleic acid modification at a specific position on a probe and specially optimized metal catalytic ions, and meanwhile, the reagent can be stably stored for 12 months at the temperature of 2-8 DEG C.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a specific probe, a primer-probe combination and a kit for detecting drug-resistant mutations of Mycoplasma pneumoniae and Bordetella pertussis. Background Art

[0002] Mycoplasma pneumoniae (MP) can cause mycoplasma pneumonia, upper respiratory tract infection, bronchitis and other diseases, and is one of the most common community-acquired pneumonias. Mycoplasma pneumoniae infection can directly cause extrapulmonary infection through direct local inflammation, vascular embolism or indirect immune regulation. Due to the destruction of respiratory epithelial cells by Mycoplasma pneumoniae, Mycoplasma pneumoniae infection is often complicated and secondary to mixed infection with other pathogens. Therefore, laboratory diagnosis of Mycoplasma pneumoniae is required for other systemic diseases that may be caused by Mycoplasma pneumoniae, as well as clear bacterial or viral pneumonia.

[0003] The gold standard for determining MP resistance is to use solid culture-positive colonies to determine the minimum inhibitory concentration of antimicrobial drugs, which is difficult to carry out in clinical practice. The mechanism of MP resistance to macrolide antibiotics is mainly base mutations at sites 2063, 2064, 2067, or 2617 of the 23S rRNA gene, among which mutations at sites 2063 or 2064 can lead to high-level resistance, and base mutations at site 2617 lead to low-level resistance. Currently, the most common mutation sites in clinical practice are sites 2063 and 2064.

[0004] Pertussis is a severe acute respiratory infectious disease caused by Bordetella pertussis. It is highly contagious and the course of the disease can last for several months. The general population is susceptible, especially infants and young children. It is one of the main infectious diseases that threaten children's health and life. The typical clinical manifestations are paroxysmal spasmodic cough, cock-crow-like inspiratory echo and cyanosis. Infants and young children may also have complications such as pneumonia and encephalopathy, and may suffocate, convulse, and even endanger their lives. Before the widespread vaccination of pertussis vaccine, it was the infectious disease with the highest mortality rate in children. With the implementation of the child immunization program, the immunization coverage of diphtheria, pertussis and tetanus combined vaccine (DPT) has gradually increased, which has significantly reduced the infection rate and mortality rate of pertussis. However, according to statistics from the World Health Organization (WHO), the incidence of pertussis has been on the rise in recent years. This phenomenon is called the reappearance of pertussis. The resurgence of pertussis is a global public health issue. Atypical pertussis cases are easily missed in clinical practice, and many medical institutions have not conducted laboratory diagnosis of pertussis. At the same time, my country is also facing the clinical problem of Bordetella pertussis being highly resistant to first-line antibiotics, macrolides.

[0005] At present, laboratory diagnosis of pertussis mainly relies on etiological diagnosis, nasopharyngeal swab PCR detection and serum pertussis toxin IgG (PT-IgG) detection. Although the bacterial culture method has the highest specificity, the sensitivity varies greatly due to various factors, and the culture time is long. The method is not suitable for standardization, so the positive detection rate is low, and it is impossible to make a rapid diagnosis of pertussis, which is far from meeting clinical needs. The serological detection method mainly uses ELISA to quantitatively detect the specific IgG antibodies against pertussis toxin (PT) unique to Bordetella pertussis. It has high sensitivity and specificity, is simple and fast. However, this method is not sensitive to patients in the acute phase, and cannot make an accurate diagnosis for patients who have recently received pertussis vaccines. The nucleic acid amplification method is short in time, sensitive and specific, and can detect macrolide resistance mutation sites. It has become the optimal detection method for laboratory diagnosis of pertussis.

[0006] In recent years, there have been reports of clustered epidemics of Mycoplasma pneumoniae and pertussis. Currently, there is no detection primer probe combination and kit that can simultaneously detect Mycoplasma pneumoniae and drug-resistant mutation sites, Bordetella pertussis and drug-resistant mutation sites. Summary of the invention

[0007] In view of this, the present invention provides a specific probe, a primer-probe combination and a kit for detecting drug-resistant mutations of Mycoplasma pneumoniae and drug-resistant mutations of Bordetella pertussis. The primer-probe combination and the kit provided by the present invention can distinguish between non-mutation sensitive strains and mutant strains of Mycoplasma pneumoniae and can distinguish between non-mutation sensitive strains and mutant strains of Bordetella pertussis, the detection sensitivity can reach 25 copies / mL, and has good reagent stability.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides specific probes, including specific probe 1 for detecting amplified products of Mycoplasma pneumoniae and / or specific probe 2 for detecting amplified products of Bordetella pertussis; the specific probe 1 has a nucleotide sequence as shown in SEQ ID NO:29, and at least one base among the 7th, 11th, and 12th bases at the 5' end of the specific probe is modified with a locked nucleic acid; the specific probe 2 has a nucleotide sequence as shown in SEQ ID NO:30, and at least one base among the 7th, 11th, and 15th bases at the 5' end of the specific probe is modified with a locked nucleic acid.

[0010] In some specific embodiments of the present invention, the nucleotide sequence of the specific probe 1 is: CGGGGTCnnTCCGTC (SEQ ID NO: 29), wherein n is selected from T and C and is not C at the same time.

[0011] The nucleotide sequence of the specific probe 2 is: TGGGGTCTTnCCGTCT (SEQ ID NO: 30), wherein n is selected from T and C.

[0012] The present invention provides a primer-probe combination, including a primer-probe combination 1, a primer-probe combination 2, a primer-probe combination 3, a primer-probe combination 4 and / or a primer-probe combination 5;

[0013] (I), the primer-probe combination 1 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 3;

[0014] The primer-probe combination 2 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 7;

[0015] The primer-probe combination 3 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 9;

[0016] The primer-probe combination 4 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 12, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 13, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 15;

[0017] The primer-probe combination 5 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 12, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 13, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 17;

[0018] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0019] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0020] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (I), (II) or (III).

[0021] In some specific embodiments of the present invention, the 5' end of the specific probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

[0022] In some specific embodiments of the present invention, the fluorescent groups include FAM, HEX, ROX, CY5, VIC, and JOE; the quenching groups include BHQ1, BHQ2, BHQ3, SQ1, and SQ2.

[0023] Preferably, the 5' end of the specific probe having the nucleotide sequence shown in SEQ ID NO: 3 is modified with a FAM group, and the 3' end is modified with a BHQ1 group;

[0024] The 5' end of the specific probe having the nucleotide sequence shown in SEQ ID NO: 7 is modified with a HEX group, and the 3' end is modified with a BHQ1 group;

[0025] The 5' end of the specific probe having the nucleotide sequence shown in SEQ ID NO: 9 is modified with a HEX group, and the 3' end is modified with a BHQ1 group;

[0026] The 5' end of the specific probe having the nucleotide sequence shown in SEQ ID NO: 15 is modified with a ROX group, and the 3' end is modified with a BHQ2 group;

[0027] The specific probe having the nucleotide sequence shown in SEQ ID NO. 17 has a CY5 group modified at its 5' end and a BHQ2 group modified at its 3' end.

[0028] The present invention also provides the use of the primer-probe combination in any of the following items:

[0029] (I) preparing a reagent or kit for detecting drug resistance of Mycoplasma pneumoniae; and / or

[0030] (II), preparing a reagent or kit for detecting drug-resistant mutations of Mycoplasma pneumoniae; and / or

[0031] (III) preparing a reagent or kit for detecting wild-type Mycoplasma pneumoniae and / or drug-resistant Mycoplasma pneumoniae; and / or

[0032] (IV) preparing a reagent or kit for detecting drug resistance of Bordetella pertussis; and / or

[0033] (V) preparing a reagent or kit for detecting drug-resistant mutations of Bordetella pertussis; and / or

[0034] (VI) preparing a reagent or a kit for detecting wild-type Bordetella pertussis and / or drug-resistant Bordetella pertussis.

[0035] In some specific embodiments of the present invention, the Mycoplasma pneumoniae drug-resistant mutation includes mutations at positions 2063 and 2064 of the Mycoplasma pneumoniae 23srRNA gene; the Bordetella pertussis drug-resistant mutation includes mutations at position 2047 of the Bordetella pertussis 23s rRNA gene.

[0036] The present invention also provides a reagent comprising the primer-probe combination.

[0037] The present invention also provides a kit, comprising any of the following items, and acceptable adjuvants or auxiliary agents:

[0038] (I), the primer-probe combination; and / or

[0039] (II), the reagent.

[0040] In some specific embodiments of the present invention, the kit further comprises one or more of dNTP, DNA polymerase, buffer, and enzyme activator;

[0041] The buffer comprises one or more of Tricine, KOAc, glycerol and DMSO.

[0042] In some specific embodiments of the present invention, the enzyme activator comprises manganese acetate and / or magnesium chloride.

[0043] In some specific embodiments of the present invention, the final concentration of manganese acetate is 3.5-4.5 mmol / μL; the final concentration of magnesium chloride is 3.5-4.5 mmol / μL.

[0044] In some specific embodiments of the present invention, the molar ratio of manganese acetate to magnesium chloride is 1:1.

[0045] In some specific embodiments of the present invention, the reaction system of the kit comprises:

[0046]

[0047] The present invention also provides a detection method for detecting a sample to be tested based on any of the following items:

[0048] (I), the primer-probe combination; and / or

[0049] (II), the reagent; and / or

[0050] (III) the kit.

[0051] In some specific embodiments of the present invention, the detection method comprises the following steps:

[0052] Step 1, take the sample to be tested and extract DNA;

[0053] Step 2: using the primer-probe combination, the reagent or the kit to perform PCR amplification to obtain a Ct value; if the Ct value is <40, the result is determined to be positive; otherwise, the result is determined to be negative.

[0054] In some specific embodiments of the present invention, the reaction procedure of the PCR amplification includes: 94-96°C for 2 min, 94-96°C for 10 s, 59-61°C for 22 s, and 45 cycles of amplification.

[0055] The present invention provides the following beneficial effects:

[0056] The present invention provides a primer-probe combination and a kit that can simultaneously detect Mycoplasma pneumoniae and macrolide resistance mutation sites, Bordetella pertussis and macrolide resistance mutation sites, which are special in that they can distinguish Mycoplasma pneumoniae non-mutation sensitive strains and 23s rRNA gene 2063, 2064 site mutations and Bordetella pertussis non-mutation sensitive strains and 23s rRNA gene 2047 site mutations, with high detection sensitivity, Mycoplasma pneumoniae sensitive strains, drug-resistant mutant strains and Bordetella pertussis sensitive strains, drug-resistant mutant strains sensitivity can reach 25 copies / mL, and by using locked nucleic acid modification at specific positions on the probe and specially optimized metal catalytic ions to achieve high sensitivity and specificity, the reagent can be stably stored at 2 to 8°C for 12 months. The present invention adds magnesium chloride and manganese acetate with final concentrations of 3.5 mmol / μL to 4.5 mmol / μL, respectively, and the stability of the kit can reach 2 to 8°C for 12 months without non-specific amplification. Combined with the use of locked nucleic acid modification at specific positions of the probe, the specificity and stability of the reaction system are improved while avoiding the problem of non-specific amplification caused by high concentrations of metal catalytic ions. DETAILED DESCRIPTION

[0057] The present invention discloses specific probes, primer-probe combinations and kits for detecting drug-resistant mutations of Mycoplasma pneumoniae and Bordetella pertussis. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] The prior art has low specificity for simultaneously detecting Mycoplasma pneumoniae and macrolide resistance mutation sites, Bordetella pertussis and macrolide resistance mutation sites, and cannot be stored at 2-8°C for a long time. In view of the above problems, the present invention provides a primer-probe combination and a kit for simultaneously detecting Mycoplasma pneumoniae and macrolide resistance mutation sites, Bordetella pertussis and macrolide resistance mutation sites. The special feature of the primer-probe combination and a kit is that the primer-probe combination can distinguish Mycoplasma pneumoniae non-mutation sensitive strains and 23srRNA gene site mutations at sites 2063 and 2064, and can distinguish Bordetella pertussis non-mutation sensitive strains and 23s rRNA gene site mutations at site 2047. The detection sensitivity is high, and the sensitivity of Mycoplasma pneumoniae sensitive strains, resistant mutant strains, Bordetella pertussis sensitive strains, and resistant mutant strains can reach 25 copies / mL. In addition, by using locked nucleic acid modification at specific positions on the probe and specially optimized metal catalytic ions to achieve high sensitivity and specificity, the reagent can be stably stored at 2-8°C for 12 months. The special feature of this metal catalytic ion is the use of high-concentration magnesium chloride and manganese acetate solutions in a ratio of 1:1. Magnesium chloride and manganese acetate are used as metal catalytic ions, usually at a concentration of 1 to 3 mmol / / μL in the PCR reaction system, to activate the activity of the catalytic enzyme, in conjunction with primers, probes and other components, to complete real-time fluorescence quantitative PCR. In this reaction system, by adding magnesium chloride and manganese acetate at a final concentration of 4 mmol / μL and a ratio of 1:1, combined with the use of locked nucleic acid modification at specific positions of the probe, the specificity and stability of the reaction system are improved, while the problem of non-specific amplification caused by high-concentration metal catalytic ions does not occur.

[0059] Unless otherwise specified, the specific probes, primer-probe combinations for detecting drug-resistant mutations of Mycoplasma pneumoniae and Bordetella pertussis, and raw materials and reagents used in the kit provided by the present invention can all be purchased from the market.

[0060] The present invention will be further described below in conjunction with embodiments:

[0061] Example 1 Primer-probe combination screening and verification

[0062] 1. Instruments and reagents

[0063] Fully automatic nucleic acid purification and real-time fluorescence PCR analysis system (Antu Experimental Instrument (Zhengzhou) Co., Ltd.), nucleic acid extraction and purification reagents (Antu Nucleic Acid Extraction and Purification Reagents).

[0064] 2. Nucleic acid extraction and purification

[0065] DNA was extracted according to the instructions of the nucleic acid extraction and purification kit (manufacturer: Zhengzhou Antu Bioengineering Co., Ltd.; registration number: Yuzheng Xiebei 20180037). After extraction, the sample was placed at -80°C for use (template).

[0066] 3. Design of detection primers and probes

[0067] The genome sequences of Mycoplasma pneumoniae and pertussis were searched in the NCBI database, and primers and probes were designed for the wild type of Mycoplasma pneumoniae, the 2063 and 2064 mutation sites of the drug-resistant mutant of Mycoplasma pneumoniae, the wild type of pertussis and the 2047 drug-resistant mutation site of pertussis as shown in Table 1:

[0068] Table 1 Primer probe sequences

[0069]

[0070]

[0071] Note: LNA refers to C-base locked nucleic acid modification.

[0072] 4. Primer-probe combination screening and verification

[0073] The fully automatic nucleic acid purification and real-time fluorescence PCR analysis system was used to extract DNA from wild-type Mycoplasma pneumoniae, Mycoplasma pneumoniae 2063 / 2064 site mutation, Bordetella pertussis wild-type, Bordetella pertussis 2047 site mutation plasmid samples for fluorescence quantitative PCR. The three different probes with different locked nucleic acid modification positions of each type were verified for specificity and stability. The stability verification was accelerated at 37°C, that is, excessive conditions were used to increase the chemical or physical degradation rate of the product, so as to predict its shelf life. At the same time, positive and negative controls were set. The reaction system is shown in Table 2:

[0074] Table 2 Reaction system

[0075]

[0076]

[0077] The PCR program was run in the fully automatic nucleic acid purification and real-time fluorescence PCR analysis system as follows: 95°C for 2 min, 95°C for 10 s, and 60°C for 22 s for a total of 45 cycles.

[0078] The test results are shown in Table 3 below:

[0079] Table 3 Primer-probe combination screening and verification

[0080]

[0081]

[0082]

[0083] Based on the above results, it can be seen that for wild-type plasmid samples of Mycoplasma pneumoniae, both the combinations of MP probe 1 and MP probe 3 did not show non-specific amplification and could be detected normally. For the 2063 site mutation plasmid samples of Mycoplasma pneumoniae, both the combinations of 2063 probe 1 and 2063 probe 2 did not show non-specific amplification and could be detected normally. For the 2064 site mutation plasmid samples of Mycoplasma pneumoniae, both the combinations of 2064 probe 1 and 2064 probe 3 did not show non-specific amplification and could be detected normally. For the wild-type plasmid samples of Bordetella pertussis, both the combinations of BP probe 1 and BP probe 2 did not show non-specific amplification and could be detected normally. For the 2047 site mutation plasmid samples of Bordetella pertussis, both the combinations of 2047 probe 1 and 2047 probe 3 did not show non-specific amplification and could be detected normally. Although nonspecific amplification did not occur in the above combinations, after 7 days of acceleration at 37°C, the detection Ct value declined compared to 0 days. There may be risks in the stability of the reagents, and the reaction system needs to be further optimized to improve the stability of the reagents.

[0084] Example 2 Determination of the concentration of each component in the reaction system

[0085] In order to determine the concentration of each component of the reaction system of this kit, the concentrations of each PCR buffer, primer probe, dNTP and rTth enzyme were set as shown in Table 4. The detection ability of the reagent at different concentrations was verified by extracting DNA from wild-type Mycoplasma pneumoniae, Mycoplasma pneumoniae 2063 / 2064 site mutation, Bordetella pertussis wild-type, Bordetella pertussis 2047 site mutation plasmid samples and negative samples for fluorescent quantitative PCR.

[0086] Table 4 Reaction system

[0087]

[0088]

[0089] Table 5 Test results

[0090]

[0091]

[0092] The above test results show that when the PCR buffer, primer probe, dNTP and rTth enzyme are within the above concentration range, there is no significant difference in the test results among experimental group 1, experimental group 2 and experimental group 3.

[0093] Example 3 Primer-probe combination stability verification

[0094] In order to improve the stability of the primer-probe combination of this kit, the components of the reaction system were optimized. The specific optimization method was to adjust the concentration of manganese acetate in the reaction system and add magnesium chloride solution of the same concentration at a 1:1 ratio to improve the stability of the kit. DNA was extracted from wild-type Mycoplasma pneumoniae, Mycoplasma pneumoniae 2063 / 2064 site mutation, wild-type Bordetella pertussis, Bordetella pertussis 2047 site mutation plasmid samples and negative samples for fluorescent quantitative PCR to verify the stability and specificity of the reagent under different concentrations of metal catalytic ions. The stability assessment method adopted real-time stability verification. The reaction system and test results of the control group and each experimental group are as follows:

[0095] Table 6 Reaction system construction

[0096]

[0097]

[0098] Table 7 Stability test results

[0099]

[0100]

[0101] The above test results show that for wild-type plasmid samples of Mycoplasma pneumoniae, when the concentration of manganese acetate and magnesium chloride of MP probe 1 primer probe combination is between 3.5mmol / μL and 4.5mmol / μL, the stability of the kit can reach 2-8℃ for 12 months without non-specific amplification. For the 2063 site mutation plasmid samples of Mycoplasma pneumoniae, when the concentration of manganese acetate and magnesium chloride of 2063 probe 2 primer probe combination is between 3.5mmol / μL and 4.5mmol / μL, the stability of the kit can reach 2-8℃ for 12 months without non-specific amplification. For the 2064 site mutation plasmid samples of Mycoplasma pneumoniae, when the concentration of manganese acetate and magnesium chloride of 2064 probe 1 primer probe combination is between 3.5mmol / μL and 4.5mmol / μL, the stability of the kit can reach 2-8℃ for 12 months without non-specific amplification. For wild-type plasmid samples of Bordetella pertussis, when the concentration of manganese acetate and magnesium chloride of BP probe 2 was between 3.5mmol / μL and 4.5mmol / μL, the stability of the kit could reach 12 months at 2-8°C without non-specific amplification. For plasmid samples with mutations at site 2047 of Bordetella pertussis, when the concentration of manganese acetate and magnesium chloride of 2047 probe 1 was between 3.5mmol / μL and 4.5mmol / μL, the stability of the kit could reach 12 months at 2-8°C without non-specific amplification. Therefore, when the primer probe combination and reaction system of this kit are as shown in Table 6, this kit has good specificity and can be stably stored at 2-8°C for 12 months.

[0102] Table 8 Kit primer probe combination and reaction system

[0103]

[0104]

[0105] Example 4 Establishment of positive judgment value of the kit

[0106] The ROC curve method is used to determine the positive judgment value for each target in this kit. It calculates a series of sensitivities and specificities by setting multiple different critical values ​​for continuous variables, and then draws a curve with sensitivity as the ordinate and (1-specificity) as the abscissa. The larger the area under the curve, the higher the diagnostic accuracy. That is, assuming the sensitivity and specificity of the test under different positive judgment values, the point with the best sensitivity and specificity is used as the positive judgment value. The test results of this kit are expressed as Ct values, and the determination of the target positive judgment value is the determination of the Ct value with the best sensitivity and specificity.

[0107] The positive judgment values ​​of wild-type Mycoplasma pneumoniae, drug-resistant mutations of Mycoplasma pneumoniae 2063 and 2064, wild-type pertussis, and drug-resistant mutations of pertussis 2047 determined by this kit are all 40. The specific test results are analyzed as follows:

[0108] Table 9 Explanation of test results

[0109]

[0110] Example 5 Determination of the detection limit of the kit

[0111] Five different concentrations of Mycoplasma pneumoniae wild-type samples and various drug-resistant mutant samples, Bordetella pertussis wild-type and A2047G drug-resistant mutant samples were selected. Each concentration sample was tested 20 times in duplicate, and the detection rate of each concentration was counted. The test results are as follows:

[0112] Table 10 Confirmation of the detection limit of wild type Mycoplasma pneumoniae

[0113] Sample concentration (copies / mL) Detection of repeat count Number of positive tests Positive detection rate 3 20 0 0.00％ 6 20 6 30.00％ 12.5 20 18 90.00％ 25 20 20 100.00％ 50 20 20 100.00％

[0114] Table 11 Confirmation of detection limit of Mycoplasma pneumoniae A2063G resistant mutant

[0115] Sample concentration (copies / mL) Detection of repeat count Number of positive tests Positive detection rate 3 20 0 0.00％ 6 20 9 45.00％ 12.5 20 17 85.00％ 25 20 20 100.00％ 50 20 20 100.00％

[0116] Table 12 Confirmation of detection limit of Mycoplasma pneumoniae A2064G resistant mutant

[0117] Sample concentration (copies / mL) Detection of repeat count Number of positive tests Positive detection rate 3 20 0 0.00％ 6 20 11 55.00％ 12.5 20 17 85.00％ 25 20 20 100.00％ 50 20 20 100.00％

[0118] Table 13 Confirmation of detection limit of wild type pertussis

[0119] Sample concentration (copies / mL) Detection of repeat count Number of positive tests Positive detection rate 3 20 0 0.00％ 6 20 10 50.00％ 12.5 20 15 75.00％ 25 20 20 100.00％ 50 20 20 100.00％

[0120] Table 14 Confirmation of detection limit of pertussis A2047G drug-resistant mutant

[0121] Sample concentration (copies / mL) Detection of repeat count Number of positive tests Positive detection rate 3 20 0 0.00％ 6 20 11 55.00％ 12.5 20 17 85.00％ 25 20 20 100.00％ 50 20 20 100.00％

[0122] The above test results show that the detection sensitivity (LOD) of the detection method of this kit for sensitive strains and resistant mutant strains of Mycoplasma pneumoniae and sensitive strains and resistant mutant strains of Bordetella pertussis can reach 25 copies / mL.

[0123] Example 6 Pathogen Cross-Reaction Verification

[0124] This kit was used to detect influenza A virus, influenza B virus, metapneumovirus, parainfluenza virus, syncytial virus, adenovirus, Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, Acinetobacter baumannii, Klebsiella pneumoniae, Burkholderia cepacia, Legionella pneumophila, Escherichia coli, Staphylococcus epidermidis, Chlamydia pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae and other pathogens to verify the cross-reaction of this kit to other pathogens. The test results are as follows:

[0125] Table 15 Cross-pathogen detection

[0126]

[0127] The above test results show that this kit has no cross reaction with samples such as influenza A virus, influenza B virus, metapneumovirus, parainfluenza virus, syncytial virus, adenovirus, Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, Acinetobacter baumannii, Klebsiella pneumoniae, Burkholderia cepacia, Legionella pneumophila, Escherichia coli, Staphylococcus epidermidis, Chlamydia pneumoniae, Pseudomonas aeruginosa, and Haemophilus influenzae.

[0128] Example 7 Anti-interference to potential exogenous substances

[0129] The two concentration values ​​of wild type Mycoplasma pneumoniae and A2063G / A2064G drug-resistant mutants were selected, and the two concentration values ​​of wild type Bordetella pertussis and A2047G drug-resistant mutants were selected. The two concentration values ​​were 1×10 5 At the same time, add the interfering substances to the corresponding samples at 3 times the peak concentration (Cmax), and calculate the interference rate by the interference rate calculation formula: (interference sample concentration - control sample concentration) / control sample concentration × 100%. If the interference rate % is less than the correctness bias allowed by the project standard (set to 10%), it can be judged as passed. The specific test results are as follows:

[0130] Table 16 Anti-interference experiment of exogenous substances

[0131] Drug Name Interference rate (%) Drug Name Interference rate (%) Phenylephrine 2.1 Ribavirin 3.1 Dexamethasone 2.7 Oseltamivir 2.7 Budesonide 3.5 Peramivir 2.9 Zanamivir 2.1 Mupirocin 3.5 Oxymetazoline 1.9 Tobramycin 2.3

[0132] The above experiments show that when the sample contains common drugs such as phenylephrine, dexamethasone, budesonide, zanamivir, oxymetazoline, ribavirin, oseltamivir, peramivir, mupirocin, and tobramycin, the detection sensitivity of the kit of the present invention will not be significantly disturbed.

[0133] Example 8 Anti-interference to potential endogenous substances

[0134] The two concentration values ​​of wild type Mycoplasma pneumoniae and A2063G / A2064G drug-resistant mutants were selected, and the two concentration values ​​of wild type Bordetella pertussis and A2047G drug-resistant mutants were selected. The two concentration values ​​were 1×10 5 At the same time, the interfering substances blood and mucin were added to the corresponding samples at a ratio of 2% v / v and 5% v / v respectively. The interference rate was calculated by the interference rate calculation formula: (interference sample concentration - control sample concentration) / control sample concentration × 100%. If the interference rate % is less than the correctness bias allowed by the project's line standard (set to 10%), it can be judged as passed. The specific test results are as follows:

[0135] Table 17 Anti-interference experiment of potential endogenous substances

[0136] Interfering substances Interference rate (%) blood 3.6 Mucin 2.4

[0137] The above experiments show that when the sample contains blood (2%) and mucin (5%), the detection sensitivity of the kit of the present invention will not be significantly disturbed.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A specific probe, characterized in that It comprises a specific probe 1 for detecting amplified products of Mycoplasma pneumoniae and / or a specific probe 2 for detecting amplified products of Bordetella pertussis; the specific probe 1 has a nucleotide sequence as shown in SEQ ID NO:29, and at least one base among the 7th, 11th and 12th bases at the 5' end of the specific probe is modified with a locked nucleic acid; the specific probe 2 has a nucleotide sequence as shown in SEQ ID NO:30, and at least one base among the 7th, 11th and 15th bases at the 5' end of the specific probe is modified with a locked nucleic acid.

2. A primer-probe combination, characterized in that: Including any one or more of primer probe combination 1 for detecting wild type of Mycoplasma pneumoniae, primer probe combination 2 for detecting A2063G variant of Mycoplasma pneumoniae, primer probe combination 3 for detecting A2064G variant of Mycoplasma pneumoniae, primer probe combination 4 for detecting wild type of Bordetella pertussis, or primer probe combination 5 for detecting A2047G variant of Bordetella pertussis; (I), the primer-probe combination 1 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 3; The primer-probe combination 2 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 7; The primer-probe combination 3 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 9; The primer-probe combination 4 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 12, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 13, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 15; The primer-probe combination 5 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 12, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 13, and a specific probe having a nucleotide sequence as shown in SEQ ID NO: 17; (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (I), (II) or (III).

3. Use of the specific probe according to claim 1 or the primer-probe combination according to claim 2 in any of the following: (I) preparing a reagent or kit for detecting drug resistance of Mycoplasma pneumoniae; and / or (II), preparing a reagent or kit for detecting drug-resistant mutations of Mycoplasma pneumoniae; and / or (III) preparing a reagent or kit for detecting wild-type Mycoplasma pneumoniae and / or drug-resistant Mycoplasma pneumoniae; and / or (IV), preparing a reagent or a kit for detecting drug resistance of Bordetella pertussis; and / or (V) preparing a reagent or kit for detecting drug-resistant mutations of Bordetella pertussis; and / or (VI) preparing a reagent or a kit for detecting wild-type Bordetella pertussis and / or drug-resistant Bordetella pertussis.

4. The use according to claim 3, characterized in that The Mycoplasma pneumoniae drug-resistant mutation includes mutations at positions 2063 and 2064 of the Mycoplasma pneumoniae 23s rRNA gene; the Bordetella pertussis drug-resistant mutation includes mutations at position 2047 of the Bordetella pertussis 23s rRNA gene.

5. A reagent, characterized in that It comprises the specific probe as claimed in claim 1 or the primer-probe combination as claimed in claim 2.

6. A kit, characterized in that Include any of the following, and acceptable excipients or adjuvants: (I), the specific probe according to claim 1 or the primer-probe combination according to claim 2; and / or (II) The reagent as described in claim 5.

7. The kit according to claim 6, characterized in that Also includes one or more of dNTP, DNA polymerase, buffer, enzyme activator; The buffer comprises one or more of Tricine, KOAc, glycerol and DMSO.

8. The kit according to claim 6 or 7, characterized in that The enzyme activator includes manganese acetate and / or magnesium chloride.

9. The kit according to any one of claims 6 to 8, characterized in that The final concentration of the manganese acetate is 3.5 to 4.5 mmol / μL; the final concentration of the magnesium chloride is 3.5 to 4.5 mmol / μL.

10. A detection method, characterized in that: Detect samples based on any of the following: (I), the specific probe according to claim 1 or the primer-probe combination according to claim 2; and / or (II), the reagent as claimed in claim 5; and / or (III) The kit according to any one of claims 6 to 9.

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