MP primer group, kit and application of MP primer group in mycoplasma pneumoniae nucleic acid detection

Abstract

The invention provides an MP primer group, a kit and application of the MP primer group in mycoplasma pneumoniae nucleic acid detection, and relates to the technical field of mycoplasma pneumoniae detection. The MP primer group comprises at least one of a first primer group and a second primer group; the first primer group consists of a pair of outer primers F3-1 and B3-1, a pair of inner primers FIP-1 and BIP-1, and a pair of loop primers LF-1 and LB-1; and the second primer group consists of a pair of outer primers F3-2 and B3-2, a pair of inner primers FIP-2 and BIP-2, and a pair of loop primers LF-2 and LB-2. According to the MP primer group provided by the invention, the specificity and the flexibility of detection are improved, the reliability is ensured, and the sensitivity is improved by increasing the amplification speed. The constant temperature characteristic of the LAMP technology reduces cross contamination, so that the detection is rapid and safe, and the LAMP technology is suitable for on-site rapid diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of Mycoplasma pneumoniae detection, and in particular to an MP primer set, a kit and applications thereof in Mycoplasma pneumoniae nucleic acid detection. Background Art

[0002] Mycoplasma pneumoniae (MP) infection is a common respiratory infection with periodic epidemic characteristics worldwide, especially in children and adolescents. Community-acquired pneumonia caused by MP infection can account for 20% to 40% during the epidemic period, with various clinical manifestations, including fever, sore throat, dry cough, etc. Some patients may develop serious complications. Due to its atypical characteristics, it is difficult to distinguish it from pneumonia caused by other causes based on clinical manifestations alone. Therefore, the development of simple, rapid and accurate MP detection methods is crucial for early warning, clinical diagnosis and treatment.

[0003] Among the new technologies for diagnosing MP infection, polymerase chain reaction (PCR) has become the preferred method for direct pathogen detection due to its high specificity and high sensitivity. PCR technology achieves accurate detection of pathogens by amplifying the nucleic acid sequence of pathogens. However, PCR-based detection methods require professionals to perform complex sample pre-processing and instrument analysis, and are heavily dependent on laboratory resources, limiting their application in rapid screening and on-site testing.

[0004] Isothermal amplification technology, especially loop-mediated isothermal amplification (LAMP) technology, provides a simple and efficient nucleic acid amplification detection solution. LAMP technology relies on specific primers and strand-displacing DNA polymerase to achieve automatic cyclic amplification of target DNA under constant temperature conditions. Combined with real-time fluorescent probe technology, it can quickly interpret the test results and has important application value for pathogen detection. However, LAMP technology also faces the challenge of sample pre-processing. It is necessary to extract and purify nucleic acids from samples, which is often cumbersome and time-consuming.

[0005] Traditional nucleic acid extraction methods, including magnetic bead method, thermal lysis method, column extraction method, etc., are generally complicated and time-consuming, which is not conducive to the rapid screening and diagnosis of MP infection. These methods not only increase the time cost of detection, but also increase the requirements for professionals and equipment, limiting the popularization and application of detection technology. In addition, the complexity of sample pretreatment may also lead to sample loss and contamination, affecting the accuracy and reliability of the test results.

[0006] In summary, the existing MP detection technology has obvious defects in sample pre-processing, detection speed, operation convenience and dependence on laboratory resources, which limit the widespread application of MP detection technology, especially in rapid screening and on-site detection. Therefore, developing an MP detection technology that can overcome these defects has important clinical significance and application prospects for improving detection efficiency and accuracy and reducing operation difficulty and cost.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide an MP primer set, a kit and its application in the detection of Mycoplasma pneumoniae nucleic acid, wherein the MP primer set comprises two sets of primer combinations for Mycoplasma pneumoniae (MP) for detection by LAMP technology. These primer sets improve the specificity and flexibility of detection, ensure reliability, and improve sensitivity by accelerating the amplification speed. The constant temperature characteristic of LAMP technology reduces cross contamination, making the detection fast, safe and suitable for rapid on-site diagnosis.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] In a first aspect, the present invention provides an MP primer set, comprising at least one of a first primer set and a second primer set;

[0011] The first primer set consists of a pair of outer primers F3-1 and B3-1, a pair of inner primers FIP-1 and BIP-1, and a pair of loop primers LF-1 and LB-1;

[0012] The second primer set consists of a pair of outer primers F3-2 and B3-2, a pair of inner primers FIP-2 and BIP-2, and a pair of loop primers LF-2 and LB-2;

[0013] The nucleotide sequence of the outer primer F3-1 is shown in SEQ ID NO.1; the nucleotide sequence of the outer primer B3-1 is shown in SEQ ID NO.2; the nucleotide sequence of the inner primer FIP-1 is shown in SEQ ID NO.3; the nucleotide sequence of the inner primer BIP-1 is shown in SEQ ID NO.4; the nucleotide sequence of the loop primer LF-1 is shown in SEQ ID NO.5; the nucleotide sequence of the loop primer LB-1 is shown in SEQ ID NO.6;

[0014] The nucleotide sequence of the outer primer F3-2 is shown in SEQ ID NO.7; the nucleotide sequence of the outer primer B3-2 is shown in SEQ ID NO.8; the nucleotide sequence of the inner primer FIP-2 is shown in SEQ ID NO.9; the nucleotide sequence of the inner primer BIP-2 is shown in SEQ ID NO.10; the nucleotide sequence of the loop primer LF-2 is shown in SEQ ID NO.11; the nucleotide sequence of the loop primer LB-2 is shown in SEQ ID NO.12.

[0015] In the second aspect, the present invention provides a housekeeping gene primer set, which is used as an internal standard for detection in conjunction with the MP primer set described in the aforementioned embodiment, and the housekeeping gene primer set consists of a pair of external primers F3-3 and B3-3, a pair of internal primers FIP-3 and BIP-3, and a pair of loop primers LF-3 and LB-3; the nucleotide sequence of the external primer F3-3 is shown in SEQ ID NO.13; the nucleotide sequence of the external primer B3-3 is shown in SEQ ID NO.14; the nucleotide sequence of the internal primer FIP-3 is shown in SEQ ID NO.15; the nucleotide sequence of the internal primer BIP-3 is shown in SEQ ID NO.16; the nucleotide sequence of the loop primer LF-3 is shown in SEQ ID NO.17; the nucleotide sequence of the loop primer LB-3 is shown in SEQ ID NO.18.

[0016] In a third aspect, the present invention provides a kit, comprising: a sample releaser, a detection device, a positive control substance and a negative control substance; the detection device is a detection chip or a PCR reaction tube.

[0017] The detection device includes the MP primer set as described in the above embodiment, and the housekeeping gene primer set as described in the above embodiment;

[0018] In an optional embodiment, the positive control is a Mycoplasma pneumoniae culture;

[0019] In an optional embodiment, the negative control is physiological saline.

[0020] In an optional embodiment, the sample release agent includes: ammonium sulfate, with a molar volume concentration of 20mM to 150mM; disodium ethylenediaminetetraacetic acid, with a molar volume concentration of 1mM to 15mM; bovine serum albumin, with a mass volume concentration of 0.2mg / mL to 6mg / mL; dithiothreitol, with a molar volume concentration of 0.01M to 1M; TritonX-100, with a mass percentage concentration of 0.5% to 3%; dimethyl sulfoxide, Tween 20 and PEG-8000 Any one of the following, with a mass percentage concentration of 0.3% to 6%; formamide, with a mass percentage concentration of 0.2% to 3%; NP40, with a mass percentage concentration of 0.2% to 2%; Proclin300 or proclin950, with a mass percentage concentration of 0.01% to 0.07%; Savantine or PVP, with a molar volume concentration of 0.01mM to 0.5mM; biological buffer, with a molar volume concentration of 20mM to 150mM; the balance is deionized water;

[0021] In an alternative embodiment, the biological buffer is Tris-HCl or Bicine-KOH.

[0022] In an optional embodiment, the detection chip includes a reaction chamber; the reaction chamber includes a Mycoplasma pneumoniae detection reaction chamber and an internal standard charge reaction chamber;

[0023] The Mycoplasma pneumoniae detection reaction chamber comprises a freeze-dried amplification detection system and the MP primer set;

[0024] The internal standard charge reaction chamber contains a freeze-dried amplification detection system and the housekeeping gene primer.

[0025] In an optional embodiment, the preparation method of the freeze-dried amplification detection system comprises:

[0026] Preparing a mother solution; wherein the mother solution includes Bst polymerase, dNTP, fluorescent dye, MgSO4, betaine, biological buffer, polyethylene glycol, sugar alcohol, trehalose and sucrose;

[0027] The mother solution is transferred to a freeze-drying device and a freeze-drying program is started to perform freeze-drying treatment to obtain the freeze-dried amplification detection system in the form of freeze-dried balls after freeze-drying treatment;

[0028] In an optional embodiment, the volume of the freeze-dried pellet is 15 μL / pellet to 23 μL / pellet.

[0029] In an optional embodiment, the biological buffer is Tris-HCl or Bicine-KOH;

[0030] In an optional embodiment, the sugar alcohol is mannitol and / or sorbitol.

[0031] In an optional embodiment, the concentration of each component in the mother solution includes: Bst polymerase, 6U / reaction to 10U / reaction; dNTP, molar volume concentration of 0.5mM to 1mM; fluorescent dye, molar volume concentration of 0.5μM to 1.5μM; MgSO4, molar volume concentration of 4mM to 8mM; betaine, molar volume concentration of 0.5M to 1M; the biological buffer, molar volume concentration of 50μM to 200mM; polyethylene glycol, mass percentage concentration of 0.5% to 4%; the sugar alcohol, mass percentage concentration of 0.3% to 2%; sucrose, mass percentage concentration of 0.4% to 1.5%; trehalose, mass percentage concentration of 0.4% to 1%;

[0032] In an optional embodiment, the pH of the biological buffer is 7-9.

[0033] The polyethylene glycol is PEG8000 and / or PEG6000.

[0034] In an optional embodiment, the freeze-drying procedure includes: pre-freezing stage: set the temperature to -45°C, the duration is 120 minutes, and the vacuum degree is 0Pa; sublimation stage: set the temperature to -45°C, the duration is 180 minutes, and the vacuum degree is 20Pa; set the temperature to -40°C, the set time is 10 minutes, the duration is 720 minutes, and the vacuum degree is 20Pa; analysis stage: set the temperature to 25°C, the set time is 130 minutes, the duration is 240 minutes, and the vacuum degree is 0Pa.

[0035] In a fifth aspect, the present invention further provides a method for preparing the reaction chamber as described in the aforementioned embodiment, comprising:

[0036] The MP primer set and the housekeeping gene primer set are respectively mixed with an agarose fixative to prepare mixed primers, thereby obtaining MP mixed primers and housekeeping gene mixed primers;

[0037] Adding the MP mixed primer to the Mycoplasma pneumoniae detection reaction chamber in the reaction chamber; and adding the housekeeping gene mixed primer to the internal standard charge reaction chamber in the reaction chamber;

[0038] The reaction chamber was placed at 40° C. for drying for 30 minutes;

[0039] The dried reaction chamber is added with the freeze-dried amplification detection system in the form of freeze-dried balls and sealed.

[0040] In a sixth aspect, the present invention provides a use of a kit as described in any one of the preceding embodiments in the detection of Mycoplasma pneumoniae nucleic acid.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The MP primer set provided by the present invention comprises two sets of primer combinations designed for the conserved region of Mycoplasma pneumoniae (MP), each set consisting of an outer primer, an inner primer and a loop primer, and is used for nucleic acid detection in the loop-mediated isothermal amplification (LAMP) technology. This design improves the specificity of the detection, because both sets of primer sets can specifically identify and amplify the nucleic acid sequence of MP, reducing the amplification of non-target sequences. At the same time, providing two sets of primer combinations increases the flexibility and applicability of the detection method, and the most suitable primer set can be selected according to different experimental conditions or sample types. In addition, if one set of primer combinations fails to successfully amplify the target sequence due to mutation or contamination, the other set of primer combinations can be used as a supplement to ensure the reliability of the test results. The design of the inner primer and the loop primer helps to improve the efficiency of the LAMP reaction, accelerate the amplification speed and improve the sensitivity of the detection. The LAMP technology is carried out under constant temperature conditions, which reduces the risk of cross contamination and makes the detection safer and more reliable. Combined with the rapid amplification characteristics of the LAMP technology, the primer set can achieve rapid diagnosis of MP infection, help to take timely treatment measures, and reduce disease transmission. LAMP technology does not require complex equipment and multiple temperature cycles, making this primer set useful for rapid on-site testing, especially in areas lacking high-end laboratory equipment.

[0043] The kit provided by the present invention includes a sample releaser, a detection chip, a positive control substance and a negative control substance. After the nasal / pharyngeal swab is eluted and lysed by the sample releaser and added to the reaction chamber of the detection chip, the detection result can be judged by the fluorescence change of the reaction mixture.

[0044] The sample release agent in the kit provided in the present invention can elute and lyse the contents of the swab for downstream detection, thereby avoiding the cumbersome nucleic acid extraction process and improving the detection efficiency.

[0045] The freeze-dried amplification detection system of the detection chip in the kit provided by the present invention is prepared by freeze-drying the reaction system to obtain freeze-dried balls, which can be transported and stored at room temperature. The freeze-dried balls can be embedded in the detection chip or PCR reaction tube, and only the detection template needs to be added for detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1This is a schematic diagram of the reaction chamber of the detection chip in Example 1 of the present application;

[0048] Figure 2 This is a graph showing the test results of the positive control substance in Example 2 of the present application using the detection kit A;

[0049] Figure 3 This is a graph showing the test results of the negative control substance in Example 2 of the present application using the detection kit A;

[0050] Figure 4 This is a graph showing the test results of the positive control substance in Example 2 of the present application using the detection kit B;

[0051] Figure 5 This is a graph showing the test results of the negative control substance in Example 2 of the present application using the detection kit B;

[0052] Figure 6 This is a graph showing the test results of the MP nucleic acid detection kit A on a clinical throat swab sample (sample 1) in Example 2 of the present application;

[0053] Figure 7 This is a graph showing the test results of the MP nucleic acid detection kit A on a clinical throat swab sample (sample 2) in Example 2 of the present application;

[0054] Figure 8 This is a graph showing the test results of the MP nucleic acid detection kit A on a clinical throat swab sample (sample 3) in Example 2 of the present application;

[0055] Fig. 9 This is a graph showing the test results of the MP nucleic acid detection kit A on a clinical throat swab sample (sample 4) in Example 2 of the present application;

[0056] Fig.10 This is a graph showing the test results of the MP nucleic acid detection kit A on a clinical throat swab sample (sample 5) in Example 2 of the present application;

[0057] Fig.11 This is a graph showing the test results of the MP nucleic acid detection kit B on a clinical throat swab sample (sample 6) in Example 2 of the present application;

[0058] Fig.12 This is a graph showing the test results of the MP nucleic acid detection kit B on a clinical throat swab sample (sample 7) in Example 2 of the present application;

[0059] Fig.13 This is a graph showing the test results of the MP nucleic acid detection kit B on a clinical throat swab sample (sample 8) in Example 2 of the present application;

[0060] Fig.14 This is a graph showing the test results of the MP nucleic acid detection kit B on a clinical throat swab sample (sample 9) in Example 2 of the present application;

[0061] Fig.15 This is a graph showing the test results of the MP nucleic acid detection kit B on a clinical throat swab sample (sample 10) in Example 2 of the present application;

[0062] Fig.16 This is a graph showing the specificity test results of the MP nucleic acid detection kit A on clinical samples (Ureaplasma urealyticum) in Example 3 of the present application;

[0063] Fig.17 This is a graph showing the specificity test results of the MP nucleic acid detection kit A on clinical samples (Mycoplasma hominis) in Example 3 of the present application;

[0064] Fig.18 This is a graph showing the specificity test results of the MP nucleic acid detection kit A on clinical samples (Mycoplasma genitalium) in Example 3 of the present application;

[0065] Fig.19 This is a graph showing the specificity test results of the MP nucleic acid detection kit A on clinical samples (Chlamydia pneumoniae) in Example 3 of the present application;

[0066] Fig. 20 This is a graph showing the specificity test results of the MP nucleic acid detection kit A on clinical samples (influenza A virus) in Example 3 of the present application;

[0067] Fig.21 This is a graph showing the specificity test results of the MP nucleic acid detection kit A on clinical samples (influenza B virus) in Example 3 of the present application;

[0068] Fig. 22 This is a graph showing the specificity test results of the MP nucleic acid detection kit B on clinical samples (Ureaplasma urealyticum) in Example 3 of the present application;

[0069] Fig.23 This is a graph showing the specificity test results of the MP nucleic acid detection kit B on clinical samples (Mycoplasma hominis) in Example 3 of the present application;

[0070] Fig.24 This is a graph showing the specificity test results of the MP nucleic acid detection kit B on clinical samples (Mycoplasma genitalium) in Example 3 of the present application;

[0071] Fig.25 This is a graph showing the specificity test results of the MP nucleic acid detection kit B on clinical samples (Chlamydia pneumoniae) in Example 3 of the present application;

[0072] Fig.26 This is a graph showing the specificity test results of the MP nucleic acid detection kit B on clinical samples (influenza A virus) in Example 3 of the present application;

[0073] Fig. 27 This is a graph showing the specificity test results of the MP nucleic acid detection kit B on clinical samples (influenza B virus) in Example 3 of the present application;

[0074] Fig.28 This is a graph showing the detection limit test results of the MP nucleic acid detection kit on MP culture (first time) in Example 4 of the present application;

[0075] Fig.29 This is a graph showing the detection limit test results of the MP nucleic acid detection kit on MP culture (second time) in Example 4 of the present application;

[0076] Fig.30 This is a graph showing the detection limit test results of the MP nucleic acid detection kit on MP culture (third time) in Example 4 of the present application;

[0077] Fig.31 This is a graph showing the detection limit test results of the MP nucleic acid detection kit on MP culture (4th time) in Example 4 of the present application;

[0078] Fig.32 This is a graph showing the detection limit test results of the MP nucleic acid detection kit on MP culture (5th time) in Example 4 of the present application. DETAILED DESCRIPTION

[0079] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0080] An embodiment of the present application provides an MP primer set, comprising at least one of a first primer set and a second primer set.

[0081] The first primer set consists of a pair of outer primers F3-1 and B3-1, a pair of inner primers FIP-1 and BIP-1, and a pair of loop primers LF-1 and LB-1.

[0082] The second primer set consists of a pair of outer primers F3-2 and B3-2, a pair of inner primers FIP-2 and BIP-2, and a pair of loop primers LF-2 and LB-2.

[0083] The MP primer set includes any one of two primer sets, namely the first primer set and the second primer set. The two primer sets can be used to detect MP. The nucleotide sequences of the two primer sets are shown in Table 1 and Table 2.

[0084] Table 1. Primers and corresponding sequences in the first primer set

[0085]

[0086] Table 2. Primers and corresponding sequences in the second primer set

[0087]

[0088] The MP primer set provided in the embodiment of the present application comprises two sets of primer combinations designed for the conservative region of Mycoplasma pneumoniae (MP), each set of which is composed of an outer primer, an inner primer and a loop primer, and is used for nucleic acid detection in the loop-mediated isothermal amplification (LAMP) technology. This design improves the specificity of the detection, because both sets of primer sets can specifically identify and amplify the nucleic acid sequence of MP, reducing the amplification of non-target sequences. At the same time, providing two sets of primer combinations increases the flexibility and applicability of the detection method, and the most suitable primer set can be selected according to different experimental conditions or sample types. In addition, if one set of primer combinations fails to successfully amplify the target sequence due to mutation or contamination, the other set of primer combinations can be used as a supplement to ensure the reliability of the test results. The design of the inner primer and the loop primer helps to improve the efficiency of the LAMP reaction, speed up the amplification speed and improve the sensitivity of the detection. The LAMP technology is carried out under constant temperature conditions, which reduces the risk of cross contamination and makes the detection safer and more reliable. Combined with the rapid amplification characteristics of the LAMP technology, the primer set can achieve rapid diagnosis of MP infection, help to take timely treatment measures, and reduce disease transmission. LAMP technology does not require complex equipment and multiple temperature cycles, making this primer set useful for rapid on-site testing, especially in areas lacking high-end laboratory equipment.

[0089] A housekeeping gene primer set is provided in an embodiment of the present application, which is used as an internal standard for detection in conjunction with the MP primer set described in the aforementioned embodiment. The housekeeping gene primer set consists of a pair of outer primers F3-3 and B3-3, a pair of inner primers FIP-3 and BIP-3, and a pair of loop primers LF-3 and LB-3.

[0090] Table 3. Primers and corresponding sequences in the housekeeping gene primer set

[0091]

[0092] A kit is provided in an embodiment of the present application, comprising: a sample release agent, a detection device, a positive control substance and a negative control substance; the detection device is a detection chip or a PCR reaction tube.

[0093] The detection device includes the MP primer set as described in the above embodiment and the housekeeping gene primer set as described in the above embodiment.

[0094] The kit for detecting nucleic acid of Mycoplasma pneumoniae based on loop-mediated isothermal amplification provided in this embodiment can rapidly elute and lyse the contents of nasal / pharyngeal swabs, and determine the results through the fluorescence change after mixing with the reaction system in the detection chip. The detection process can be completed within 30 minutes, and the detection limit can reach 4 copies / μL. There is no cross reaction with Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydia pneumoniae, influenza A virus, influenza B virus, etc., and it has the characteristics of simplicity, rapidity, high sensitivity, and strong specificity. It solves the problems of complex operation, time-consuming and labor-intensive, and dependence on professional and technical personnel in traditional nucleic acid detection of Mycoplasma pneumoniae infection, and is helpful for rapid screening, diagnosis, and prognosis evaluation of diseases caused by Mycoplasma pneumoniae infection.

[0095] In an optional embodiment, the positive control is a Mycoplasma pneumoniae culture;

[0096] In an optional embodiment, the negative control is physiological saline.

[0097] In an optional embodiment, the sample release agent is shown in the following table:

[0098] Table 4. Components and addition amounts (concentrations) of sample release agents

[0099] NO. name Concentration Type Addition amount / concentration 1 Ammonium sulfate Molar volume concentration 20mM~150mM 2 Disodium EDTA Molar volume concentration 1mM~15mM 3 BSA Mass volume concentration 0.2 mg / mL to 6 mg / mL 4 Dithiothreitol Molar volume concentration 0.01M~1M 5 TritonX-100 Mass percentage concentration 0.5％～3％ 6 Any one of dimethyl sulfoxide, Tween 20 and PEG-8000 Mass percentage concentration 0.3％～6％ 7 Formamide Mass percentage concentration 0.2％～3％ 8 NP40 Percentage concentration 0.2％～2％ 9 Proclin 300 or proclin 950 Mass percentage concentration 0.01％～0.07％ 10 Savantine or PVP Molar volume concentration 0.01mM~0.5mM 11 Tris-HCl or Bicine-KOH Molar volume concentration 20mM~150mM 12 Deionized water Mass percentage concentration margin

[0100] As mentioned above, the molar volume concentration of ammonium sulfate is 20mM to 150mM, for example, it can be 20mM, 30mM, 50mM, 80mM, 100mM, 120mM, 150mM, etc.

[0101] In the above, the molar volume concentration of disodium ethylenediaminetetraacetate is 1 mM to 15 mM; for example, it can be 1 mM, 3 mM, 5 mM, 8 mM, 10 mM, 13 mM, 15 mM, etc.

[0102] As mentioned above, the mass volume concentration of bovine serum albumin is 0.2 mg / mL to 6 mg / mL; for example, it can be 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, etc.

[0103] As mentioned above, the molar volume concentration of dithiothreitol is 0.01M to 1M; for example, it can be 0.01M, 0.05M, 0.08M, 0.1M, 0.5M, 0.8M, 1M, etc.

[0104] TritonX-100 is a nonionic surfactant used in biochemical experiments to increase the permeability of cell membranes and help dissolve and release proteins and nucleic acids in cells. Its mass percentage concentration is 0.5% to 3%. For example, it can be 0.5%, 0.8%, 1%, 2%, 3%, etc.

[0105] Any one of dimethyl sulfoxide, Tween 20 and PEG-8000 has a mass percentage concentration of 0.3% to 6%; for example, it can be 0.3%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6% and the like.

[0106] Formamide, mass percentage concentration is 0.2% to 3%. For example, it can be 0.2%, 0.5%, 0.8%, 1%, 2%, 3%, etc.

[0107] NP40, also known as Nonidet P-40, is a nonionic surfactant similar to Triton X-100, used for cell lysis and protein extraction to increase membrane permeability. The mass percentage concentration is 0.2% to 2%; for example, it can be 0.2%, 0.5%, 0.8%, 1%, 2%, etc.

[0108] Proclin300 or proclin950 is a commercial preservative, commonly used for microbial control to prevent contamination of samples or reagents. Proclin300 and Proclin950 are versions with different concentrations or formulations for different application scenarios. The mass percentage concentration is 0.01% to 0.07%; for example, it can be 0.01%, 0.02%, 0.03%, 0.05%, 0.07%, etc.

[0109] Safranin refers to "Safranin", a basic dye commonly used in biochemistry and molecular biology experiments. PVP (Polyvinylpyrrolidone) is a water-soluble polymer compound commonly used for stabilization of biological samples and as an ingredient in medicines and cosmetics. The molar volume concentration of this component is 0.01mM to 0.5mM; for example, it can be 0.01mM, 0.05mM, 0.08mM, 0.1mM, 0.3mM, 0.5mM, etc.

[0110] As mentioned above, the biological buffer can be Tris-HCl or Bicine-KOH. Tris-HCl is Tris(hydroxymethyl)aminomethane hydrochloride, which is a buffer suitable for a wide pH range, especially under neutral to slightly alkaline conditions. Bicine-KOH, Bicine is N,N-bis(2-hydroxyethyl)glycine, used in combination with potassium hydroxide (KOH) to form a Bicine-KOH buffer solution, which is suitable for experiments requiring alkaline conditions. The molar volume concentration of the biological buffer is 20mM to 150mM; for example, it can be 20mM, 30mM, 50mM, 80mM, 100mM, 120mM, 150mM, and the like.

[0111] In the sample release agent, the balance is deionized water.

[0112] The main functions of these components in the sample release agent are to help lyse cells or tissues, extract and stabilize nucleic acids, and maintain a suitable pH environment, thereby providing high-quality samples for subsequent nucleic acid testing. Through the synergistic effect of these components, the sample release agent can effectively extract nucleic acids that can be used for testing from various biological samples.

[0113] The sample release agent in the kit provided in the examples of the present application can elute and lyse the contents of the swab for downstream detection, thereby avoiding the cumbersome nucleic acid extraction process and improving detection efficiency.

[0114] In an optional embodiment, the detection chip includes a reaction chamber; the reaction chamber includes a Mycoplasma pneumoniae detection reaction chamber and an internal standard charge reaction chamber. The Mycoplasma pneumoniae detection reaction chamber contains a freeze-dried amplification detection system and the MP primer set; the internal standard charge reaction chamber contains a freeze-dried amplification detection system and the housekeeping gene primer.

[0115] In an optional embodiment, the preparation method of the freeze-dried amplification detection system comprises:

[0116] Step S1, preparing a mother solution; wherein the mother solution includes Bst polymerase, dNTP, fluorescent dye, MgSO4, betaine, biological buffer, polyethylene glycol, sugar alcohol, trehalose and sucrose.

[0117] In the above steps, it is necessary to prepare a mother solution containing multiple key components, which are necessary for loop-mediated isothermal amplification (LAMP) reaction. The specific components and functions of the mother solution are as follows: Bst polymerase is a thermostable DNA polymerase that can perform DNA amplification under constant temperature conditions. Its activity range is 6-10U / reaction, providing sufficient catalytic activity to drive the LAMP reaction. dNTP contains four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), which are raw materials for DNA synthesis. Fluorescent dyes include but are not limited to Evagreen, SYBR Green, Syto, etc., which are used to monitor the changes in fluorescent signals during DNA amplification in real time, thereby achieving quantitative analysis of target nucleic acids. MgSO4 provides magnesium ions, which are essential for the activity of Bst polymerase, help stabilize the DNA polymerase-DNA complex, and promote DNA synthesis. Betaine is an organic osmotic agent that helps to improve the thermal stability and activity of the enzyme. Tris-HCl is a commonly used buffer used to maintain pH stability in the reaction system. Polyethylene glycol (PEG8000 / PEG6000) is used to increase the viscosity of the solution, which helps to improve the amplification efficiency and stability. The sugar alcohol can be at least one of mannitol and sorbitol. The sugar alcohol (such as mannitol and / or sorbitol) acts as an osmotic stabilizer to help protect the enzyme and DNA from thermal damage. Sucrose, as a carbon source, helps to stabilize enzyme activity; in addition, other substances can be used instead of sucrose as a carbon source, for example, including but not limited to maltose, a mixture of maltose and sucrose, etc. Trehalose is a non-reducing disaccharide that helps to protect the protein structure and improve thermal stability.

[0118] Step S2, transferring the mother solution to a freeze-drying device and starting a freeze-drying program to perform freeze-drying treatment to obtain the freeze-dried amplification detection system in the form of freeze-dried balls after freeze-drying treatment.

[0119] As mentioned above, in this step, the mother solution prepared in step S1 is freeze-dried for storage and transportation while maintaining the stability of its active ingredients. Transfer the mother solution to the freeze-drying equipment: transfer the prepared mother solution to a container suitable for freeze-drying and prepare for the freeze-drying procedure. Start the freeze-drying procedure: start the freeze-drying process according to the specific freeze-drying equipment and procedure. The freeze-drying procedure usually includes pre-freezing, sublimation and analysis stages to ensure that the water in the mother solution is effectively removed while maintaining the stability of the active ingredients. Obtain freeze-dried balls: after freeze-drying, the water in the mother solution is removed to form a freeze-dried amplification detection system in the form of freeze-dried balls. This form is convenient for storage and transportation at room temperature. When needed, only template DNA and water need to be added to quickly restore activity and perform LAMP reaction.

[0120] Through these two steps, a ready-to-use lyophilized amplification detection system can be effectively prepared, which is suitable for rapid on-site detection or nucleic acid detection in resource-limited environments.

[0121] The freeze drying equipment can be a freeze dryer. The mother liquid can be first placed in a liquid nitrogen bead machine to freeze and settle quickly, and then quickly transferred to the freeze dryer to run the freeze drying program to prepare freeze-dried balls.

[0122] The freeze-dried amplification detection system of the detection chip in the kit provided by the present invention is prepared by freeze-drying the reaction system to obtain freeze-dried balls, which can be transported and stored at room temperature. The freeze-dried balls can be embedded in the detection chip or PCR reaction tube, and only the detection template needs to be added for detection.

[0123] In an optional embodiment, the volume of the lyophilized pellet is 15 μL / pellet to 23 μL / pellet. For example, the volume may be 15 μL / pellet, 16 μL / pellet, 18 μL / pellet, 20 μL / pellet, 21 μL / pellet, 22 μL / pellet, 23 μL / pellet, and the like.

[0124] In an optional embodiment, the concentration of each component in the mother liquor is as shown in Table 5.

[0125] Table 5. Components and addition amounts (concentrations) in mother liquor

[0126] NO. name Concentration Type Addition amount / concentration 1 Bst polymerase The number of Bst polymerase activity units used in each reaction system 6-10U / reaction 2 dNTP Molar volume concentration 0.5mM~1mM 3 Fluorescent dyes Molar volume concentration 0.5μM~1.5μM 4 <![CDATA[MgSO4]]> Molar volume concentration 4mM~8mM 5 Betaine Molar volume concentration 0.5M~1M 6 Biological buffers Molar volume concentration 50μM~200mM 7 Polyethylene glycol Mass percentage concentration 0.5％～4％ 8 Sugar alcohols Mass percentage concentration 0.3％～2％ 9 Sucrose and / or maltose Mass percentage concentration 0.4％～1.5％ 10 Trehalose Mass percentage concentration 0.4％～1％

[0127] In an alternative embodiment, the pH of the biological buffer is 7 to 9. For example, the pH may be 7, 8, 9, etc. The polyethylene glycol is PEG8000 and / or PEG6000.

[0128] In an optional embodiment, the freeze-drying procedure includes: (1) a pre-freezing stage: the temperature is set to -45°C, the duration is 120 min, and the vacuum degree is 0 Pa; (2) a sublimation stage: the temperature is set to -45°C, the duration is 180 min, and the vacuum degree is 20 Pa; (3) the temperature is set to -40°C, the time is set to 10 min, the duration is 720 min, and the vacuum degree is 20 Pa; (4) a decomposition stage: the temperature is set to 25°C, the time is set to 130 min, the duration is 240 min, and the vacuum degree is 0 Pa.

[0129] The present application also provides a method for preparing the reaction chamber as described in the above embodiment, including:

[0130] Step S100, the MP primer set and the housekeeping gene primer set are respectively mixed with an agarose fixative to prepare mixed primers, thereby obtaining MP mixed primers and housekeeping gene mixed primers.

[0131] Step S200, adding the MP mixed primer to the Mycoplasma pneumoniae detection reaction chamber in the reaction chamber; and adding the housekeeping gene mixed primer to the internal standard charge reaction chamber in the reaction chamber.

[0132] Step S300, placing the reaction chamber at 40°C for drying for 30 minutes.

[0133] Step S400, adding the freeze-dried amplification detection system in the form of freeze-dried balls after freeze-drying into the dried reaction chamber and sealing it.

[0134] The present application provides an application of a kit as described in any of the above embodiments in the detection of Mycoplasma pneumoniae nucleic acid. In addition, it may also include: the application of the MP primer set and the housekeeping gene primer set as described in the above embodiments provided in the present application in the preparation of Mycoplasma pneumoniae nucleic acid detection products, and the detection products may include a kit (which may include detection reagents and consumables), a diagnostic reagent combination (detection reagent composition, etc.), a detection device (detection reagents, detection consumables and detection equipment), etc.

[0135] It should be noted that the kit provided in the examples of the present application and the application of the kit in the detection of Mycoplasma pneumoniae nucleic acid are not used to diagnose the disease, but as a research tool for detecting the presence of Mycoplasma pneumoniae nucleic acid in a sample. The application of the kit is intended to provide a fast and simple detection method to assist researchers and medical professionals in evaluating MP infection in research and clinical practice. The application in the examples of the present application does not replace the clinical judgment of professional medical personnel, nor should it be used as the sole basis for disease diagnosis. The use of the kit should follow the corresponding medical guidelines and regulations, and be combined with the overall clinical condition of the patient.

[0136] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0137] Example 1

[0138] In this example, a kit for detecting Mycoplasma pneumoniae nucleic acid based on loop-mediated isothermal amplification was prepared.

[0139] Experimental methods:

[0140] The kit prepared in this example includes four parts, namely, a sample release agent, a detection chip, a positive control substance, and a negative control substance.

[0141] 1. Sample release agent: 80mM ammonium sulfate, 5mM EDTA-2Na, 0.6mg / mL BSA, 0.2M DTT, 1% TritonX-100, 2% DMSO, 1% formamide, 0.8% NP40, 0.04% proclin300, 0.08mM Savantine, 100mM Tris-HCl (pH 8.8), the balance is deionized water, prepared at room temperature, no special operating environment required.

[0142] 2. Detection chip: comprising an MP detection reaction chamber and an internal standard quality control reaction chamber, wherein the MP reaction chamber comprises a freeze-dried amplification system and an MP primer set, and the internal standard quality control reaction chamber comprises a freeze-dried amplification detection system and a housekeeping gene primer.

[0143] (1) The freeze-dried amplification detection system and its preparation process are as follows: 1) The freeze-dried amplification detection system is 0.32U / μL Bst polymerase, 0.7mM dNTP, 6mM MgSO4, 1.25μM fluorescent dye Evagreen, 2% polyethylene glycol, 800mM betaine, 1% mannitol, 0.8% sucrose, 0.6% trehalose, 100mM Tris-HCl (pH 8.8), prepared at room temperature, without the need for a special operating environment. 2) After the freeze-dried amplification detection system is prepared, it is transferred to a bead machine for quick freezing and sedimentation, and the bead volume is set to 22.07μL. After the bead is dropped, it is quickly moved to the freeze dryer to execute the set freeze-drying program to prepare freeze-dried balls for use.

[0144] (2) The freeze-drying procedure is as follows: pre-freezing stage: set the temperature to -45°C, the duration is 120 min, and the vacuum degree is 0 Pa; sublimation stage: set the temperature to -45°C, the duration is 180 min, and the vacuum degree is 20 Pa; set the temperature to -40°C, the set time is 10 min, the duration is 720 min, and the vacuum degree is 20 Pa; analysis stage: set the temperature to 25°C, the set time is 130 min, the duration is 240 min, and the vacuum degree is 0 Pa.

[0145] (3) The MP reaction chamber and the internal standard charge reaction chamber are prepared as follows:

[0146] 1) The first primer set (Kit A) or the second primer set (Kit B) in the MP primer set, and the housekeeping gene primers were mixed with 0.5 μL of 0.1% agarose to prepare 2 μL of mixed primers, and the remainder was deionized water. The primers were prepared at the following concentrations: outer primers: F3 and B3, 2.5 μM; inner primers: FIP and BIP, 10 μM; loop primers: LF and LB, 5 μM.

[0147] 2) Take out the chip and place it on the table in a drying room (humidity ≤ 30%). Figure 1 As shown), 2 μL of housekeeping gene mixed primers are added to reaction well 2, and 2 μL of Mycoplasma pneumoniae mixed primers are added to reaction well 3. The chip is then placed in an oven at 40°C for 30 min. After drying, the chip is transferred to a glove box (humidity ≤ 5%), the static eliminator is turned on, and the prepared freeze-dried balls are added to reaction wells 2 and 3, respectively, and the chip is packaged by a sealing film.

[0148] 3. The positive control is the culture of Mycoplasma pneumoniae.

[0149] 4. The negative control is normal saline.

[0150] Through the above method and preparation parameters, kit A using the first primer set as the MP primer set and kit B using the second primer set as the MP primer set were finally prepared respectively.

[0151] Example 2

[0152] In this example, the prepared Mycoplasma pneumoniae detection kit based on loop-mediated isothermal amplification was used to investigate the detection performance of the kit for MP in clinical samples.

[0153] Experimental methods:

[0154] (1) The kit is the kit prepared in Example 1.

[0155] (2) Before using the kit, use kit A and kit B to test the negative control and positive control respectively; when testing the positive control, transfer all the sample release agent into the positive control tube, dissolve and mix by inversion, and then add it to the test chip; when testing the negative control, add the negative control directly to the test chip; and test it by microfluidic chip nucleic acid constant temperature amplification analyzer (GX002). The positive control result is positive, the negative control result is negative, and the test result of kit A is as follows: Figures 2-3 The test results of kit B are shown in the attached Figures 4-5 As shown, it is proved that the detection experimental environment, personnel operation, instruments and test kits are effective and can be used for clinical sample testing.

[0156] (3) The 10 throat swab samples (samples 1 to 10) were placed in a sample release agent and mixed by inversion. They were then added to the sample loading holes of the detection chips of kits A and B for amplification reaction and tested using a microfluidic chip nucleic acid isothermal amplification analyzer (GX002).

[0157] The test results of kit A are as follows Figures 6 to 10As shown; the test results of kit B are as shown Figure 11 to Figure 15 The results were all positive, indicating that the Mycoplasma pneumoniae nucleic acid detection kit based on loop-mediated isothermal amplification prepared in this example can effectively detect Mycoplasma pneumoniae in clinical sample throat swabs.

[0158] (4) The final primer concentrations in the detection reaction are as follows: F3 and B3, 0.2 μM; FIP and BIP, 0.8 μM; LF and LB, 0.4 μM.

[0159] Example 3

[0160] In this example, the prepared kit for detecting Mycoplasma pneumoniae based on loop-mediated isothermal amplification was tested for detection specificity of the kit for MP.

[0161] Experimental methods:

[0162] (1) The kit used is the kit prepared in Example 1 (Kit A and Kit B).

[0163] (2) The test environment, personnel operation, instruments, and kit effectiveness test operation are consistent with step (2) in Example 2.

[0164] (3) Samples to be tested (6 samples, each sample was tested once with kit A and once with kit B): Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydia pneumoniae, influenza A virus, and influenza B virus.

[0165] (3) Methods:

[0166] A. Take 200 μL of 1×10 6 copies / mL of the sample to be tested was added to 400 μL of sample release agent for lysis;

[0167] B. After cleavage, add the resultant to the detection chip for amplification reaction;

[0168] C. Detection was performed using a microfluidic chip nucleic acid constant temperature amplification analyzer (GX002).

[0169] Experimental results: The test results were all negative. The test results of kit A are shown in the figure and the test results of kit B are shown in the figure. Figures 16 to 21 As shown; the test results of kit B are as shown Figure 22 to Figure 27 shown.

[0170] Example 4

[0171] In this example, the prepared kit for detecting Mycoplasma pneumoniae based on loop-mediated isothermal amplification was used to investigate the minimum detection limit of the kit for MP.

[0172] Experimental methods:

[0173] (1) The kit used is the kit prepared in Example 1 (kit A).

[0174] (2) The test environment, personnel operation, instruments, and kit effectiveness test operation are consistent with step (2) in Example 2.

[0175] (3) Methods:

[0176] A. Take a throat swab from a healthy person and add MP culture to the sample release agent;

[0177] B. Dilute the MP culture to 4 copies / μL;

[0178] C. Take 200 μL of the diluted sample to be tested and add it to the detection chip for amplification reaction. Then, test it using a microfluidic chip nucleic acid constant temperature amplification analyzer (GX002). Repeat the test 5 times.

[0179] Experimental results:

[0180] Test results such as Figure 28 to Figure 32 As shown, the results were all positive, indicating that the detection limit of the Mycoplasma pneumoniae nucleic acid detection kit based on loop-mediated isothermal amplification prepared by the present invention for Mycoplasma pneumoniae is 4 copies / μL.

[0181] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An MP primer set, characterized in that: including at least one of a first primer set and a second primer set; The first primer set consists of a pair of outer primers F3-1 and B3-1, a pair of inner primers FIP-1 and BIP-1, and a pair of loop primers LF-1 and LB-1; The second primer set consists of a pair of outer primers F3-2 and B3-2, a pair of inner primers FIP-2 and BIP-2, and a pair of loop primers LF-2 and LB-2; The nucleotide sequence of the outer primer F3-1 is shown in SEQ ID NO.1; the nucleotide sequence of the outer primer B3-1 is shown in SEQ ID NO.2; the nucleotide sequence of the inner primer FIP-1 is shown in SEQ ID NO.3; the nucleotide sequence of the inner primer BIP-1 is shown in SEQ ID NO.4; the nucleotide sequence of the loop primer LF-1 is shown in SEQ ID NO.5; the nucleotide sequence of the loop primer LB-1 is shown in SEQ ID NO.6; The nucleotide sequence of the outer primer F3-2 is shown in SEQ ID NO.7; the nucleotide sequence of the outer primer B3-2 is shown in SEQ ID NO.8; the nucleotide sequence of the inner primer FIP-2 is shown in SEQ ID NO.9; the nucleotide sequence of the inner primer BIP-2 is shown in SEQ ID NO.10; the nucleotide sequence of the loop primer LF-2 is shown in SEQ ID NO.11; the nucleotide sequence of the loop primer LB-2 is shown in SEQ ID NO.

12.

2. A housekeeping gene primer set, used as an internal standard for detection in conjunction with the MP primer set of claim 1, characterized in that: The housekeeping gene primer set consists of a pair of outer primers F3-3 and B3-3, a pair of inner primers FIP-3 and BIP-3, and a pair of loop primers LF-3 and LB-3; The nucleotide sequence of the outer primer F3-3 is shown in SEQ ID NO.13; The nucleotide sequence of the outer primer B3-3 is shown in SEQ ID NO.14; The nucleotide sequence of the inner primer FIP-3 is shown in SEQ ID NO.15; The nucleotide sequence of the inner primer BIP-3 is shown in SEQ ID NO.16; The nucleotide sequence of the loop primer LF-3 is shown in SEQ ID NO.17; The nucleotide sequence of the loop primer LB-3 is shown in SEQ ID NO.

18.

3. A kit, characterized in that: include: A sample release agent, a detection device, a positive control substance and a negative control substance; the detection device is a detection chip or a PCR reaction tube; The detection device comprises the MP primer set as claimed in claim 1 and the housekeeping gene primer set as claimed in claim 2; Preferably, the positive control is a culture of Mycoplasma pneumoniae; Preferably, the negative control substance is physiological saline.

4. The kit according to claim 3, characterized in that The sample release agent comprises: Ammonium sulfate, molar volume concentration of 20mM to 150mM; Disodium ethylenediaminetetraacetate, molar volume concentration of 1mM to 15mM; Bovine serum albumin, mass volume concentration is 0.2mg / mL to 6mg / mL; Dithiothreitol, molar volume concentration of 0.01M to 1M; TritonX-100, mass percentage concentration is 0.5% to 3%; Any one of dimethyl sulfoxide, Tween 20 and PEG-8000, with a mass percentage concentration of 0.3% to 6%; Formamide, mass percentage concentration is 0.2% to 3%; NP40, mass percentage concentration is 0.2% to 2%; Proclin300 or proclin950, mass percentage concentration is 0.01% to 0.07%; Savantine or PVP, molar volume concentration is 0.01mM to 0.5mM; Biological buffer, molar volume concentration of 20mM to 150mM; The balance is deionized water; Preferably, the biological buffer is Tris-HCl or Bicine-KOH.

5. The kit according to claim 3, characterized in that The detection chip includes a reaction chamber; the reaction chamber includes a Mycoplasma pneumoniae detection reaction chamber and an internal standard charge reaction chamber; The Mycoplasma pneumoniae detection reaction chamber comprises a freeze-dried amplification detection system and the MP primer set; The internal standard charge reaction chamber contains a freeze-dried amplification detection system and the housekeeping gene primer.

6. The kit according to claim 5, characterized in that The preparation method of the freeze-dried amplification detection system comprises: Preparing a mother solution; wherein the mother solution includes Bst polymerase, dNTP, fluorescent dye, MgSO4, betaine, biological buffer, polyethylene glycol, sugar alcohol, trehalose and sucrose; The mother solution is transferred to a freeze-drying device and a freeze-drying program is started to perform freeze-drying treatment to obtain the freeze-dried amplification detection system in the form of freeze-dried balls after freeze-drying treatment; Preferably, the volume of the freeze-dried pellet is 15 μL / pellet to 23 μL / pellet; Preferably, the biological buffer is Tris-HCl or Bicine-KOH; Preferably, the sugar alcohol is mannitol and / or sorbitol.

7. The kit according to claim 6, characterized in that The concentrations of the components in the mother liquor include: Bst polymerase, 6U / reaction to 10U / reaction; dNTP, molar volume concentration 0.5mM~1mM; Fluorescent dye, molar volume concentration 0.5μM~1.5μM; MgSO4, molar volume concentration 4mM~8mM; Betaine, molar volume concentration 0.5M~1M; The biological buffer has a molar volume concentration of 50 μM to 200 mM; Polyethylene glycol, with a mass percentage concentration of 0.5% to 4%; The sugar alcohol has a mass percentage concentration of 0.3% to 2%; Sucrose, mass percentage concentration 0.4% to 1.5%; Trehalose, mass percentage concentration 0.4% to 1%; Preferably, the pH of the biological buffer is 7 to 9; Preferably, the polyethylene glycol is PEG8000 and / or PEG6000.

8. The kit according to claim 6, characterized in that The freeze-drying procedure includes: Pre-freezing stage: set the temperature to -45℃, the duration to 120min, and the vacuum degree to 0Pa; Sublimation stage: set the temperature to -45°C, duration to 180 minutes, vacuum degree to 20Pa; The set temperature is -40℃, the set time is 10min, the duration is 720min, and the vacuum degree is 20Pa; Analysis stage: set the temperature to 25°C, the time to 130 min, the duration to 240 min, and the vacuum degree to 0 Pa.

9. The kit according to claim 5, characterized in that The method for preparing the reaction chamber comprises: The MP primer set and the housekeeping gene primer set are respectively mixed with an agarose fixative to prepare mixed primers, thereby obtaining MP mixed primers and housekeeping gene mixed primers; Adding the MP mixed primer to the Mycoplasma pneumoniae detection reaction chamber in the reaction chamber; and adding the housekeeping gene mixed primer to the internal standard charge reaction chamber in the reaction chamber; The reaction chamber was placed at 40° C. for drying for 30 minutes; The dried reaction chamber is added with the freeze-dried amplification detection system in the form of freeze-dried balls and sealed.

10. Use of the kit according to any one of claims 3 to 9 in the detection of Mycoplasma pneumoniae nucleic acid.

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