Nucleic acid composition, detection system and detection device for rapidly and highly sensitively detecting mycoplasma pneumoniae

Abstract

The invention relates to the field of biological detection, in particular to a nucleic acid composition, a detection system and a detection device for rapidly and highly sensitively detecting mycoplasma pneumoniae. The nucleic acid composition comprises an isothermal amplification primer group; the isothermal amplification primer group is used for amplifying a plurality of repeated high-copy fragments in a mycoplasma pneumoniae genome. The detection system comprises a nucleic acid composition, a loop-mediated isothermal amplification reaction reagent and an indicator. Two types of genomes of mycoplasma pneumoniae are analyzed, consistent high-copy repeated fragments are found, and six primers are designed respectively, so that the amplification sensitivity is greatly improved. Meanwhile, a probe detection method is developed, non-specific amplification is greatly reduced, and the detection sensitivity is improved. The detection device comprises a first fixing plate and a test strip, the first fixing plate is connected with the test strip, loop-mediated isothermal amplification all-component freeze-drying balls are arranged in the first fixing plate, and full-closed visual rapid detection can be achieved only by adding nucleic acid fragments.

Description

Technical Field

[0001] The present application relates to the field of biological detection, and more specifically, to a nucleic acid composition, a detection system and a detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae. Background Art

[0002] Mycoplasma pneumoniae is a prokaryotic microorganism belonging to the genus Mycoplasma of the family Mycoplasma of the class Mollicutes. It is a major community-acquired pathogen that is susceptible to children and can also be transmitted between adults. Its clinical manifestations are mainly fever and cough, which can cause pneumonia and other life-threatening extrapulmonary complications as the disease progresses. Rapid and sensitive detection methods are urgently needed.

[0003] Since Mycoplasma pneumoniae grows slowly and has a long culture cycle, it is very difficult to identify it through in vitro isolation and culture. At present, the main detection methods for Mycoplasma pneumoniae are colloidal gold method and PCR method. The colloidal gold method is mainly based on the principle of antigen and antibody detection, and cannot quickly diagnose the patient population in the early stage. Molecular detection methods based on Mycoplasma pneumoniae nucleic acid, such as PCR technology, can be used for rapid diagnosis in the early stage with high sensitivity. Its limitation is that the instrument requirements are high, thermal cycling is required, and the time is long.

[0004] Loop-mediated isothermal amplification is a technology invented by Japanese scientists around 2000. It uses 4-6 primers to recognize the 4-6 segments of the 200bp length of the template sequence, and then achieves exponential amplification through the strand displacement of the strand displacement enzyme and then through stem-loop mediation. Loop-mediated isothermal amplification technology has simple primer design, convenient operation, and low requirements for machines. It can even be reacted in a water bath, and only needs to react at around 60°C for 45 minutes, which is very suitable for simple clinical rapid detection requirements.

[0005] Therefore, it is particularly important to develop a method that can quickly and sensitively detect Mycoplasma pneumoniae. Summary of the invention

[0006] In order to quickly and highly sensitively detect Mycoplasma pneumoniae, the present application provides a nucleic acid composition, a detection system and a detection device for quickly and highly sensitively detecting Mycoplasma pneumoniae.

[0007] In a first aspect, the present application provides a nucleic acid composition for rapid and highly sensitive detection of Mycoplasma pneumoniae, using the following technical solution:

[0008] A nucleic acid composition for rapid and highly sensitive detection of Mycoplasma pneumoniae comprises an isothermal amplification primer set; the isothermal amplification primer set is used to amplify multiple repeated high-copy fragments in the Mycoplasma pneumoniae genome.

[0009] By adopting the above technical scheme, according to the two genotyping genome sequences of Mycoplasma pneumoniae M129 type and FH type in NCBI (U00089 and CP002077), the repetitive fragment RepMp5 was analyzed and degenerate primers were designed. Each set of primers can amplify multiple repetitive fragments in a single genome of Mycoplasma pneumoniae, greatly improving the detection sensitivity.

[0010] Preferably, the isothermal amplification primer set consists of the following primers:

[0011] The upstream external primer F3 is shown in SEQ ID No. 1;

[0012] Downstream external primer B3 is shown as SEQ ID No.2;

[0013] The upstream internal primer FIP is shown as SEQ ID No. 3;

[0014] The downstream internal primer BIP is shown as SEQ ID No. 4;

[0015] The upstream circularization primer LF is shown in SEQ ID No.5;

[0016] The downstream circularization primer LB is shown as SEQ ID No.6.

[0017] In a second aspect, the present application provides a detection system for rapid and highly sensitive detection of Mycoplasma pneumoniae, which adopts the following technical solution:

[0018] A detection system for rapid and highly sensitive detection of Mycoplasma pneumoniae comprises the above-mentioned nucleic acid composition, a loop-mediated isothermal amplification reaction reagent and an indicator.

[0019] By adopting the above technical solution, loop-mediated isothermal amplification technology, the primer design is simple, the operation is convenient, the machine requirements are low, and the reaction can even be carried out in a water bath, and it only takes about 60°C to react for 45 minutes, which is very suitable for clinical simple rapid detection requirements. Therefore, combined with the primer set designed in this application, the detection requirements of Mycoplasma pneumoniae can be completed quickly and sensitively.

[0020] Preferably, the loop-mediated isothermal amplification reaction reagents include Bst2.0 DNA polymerase, inorganic pyrophosphatase, dNTP mixture, Tris-HCl, (NH4)2SO4, KCl, 0.1% Tween20 and MgSO4.

[0021] By adopting the above technical solution, Bst2.0 DNA polymerase can accelerate the reaction speed and improve the sensitivity of the reaction; inorganic pyrophosphatase can reduce the inhibition of the reaction and thus improve the sensitivity of the reaction.

[0022] Preferably, the indicator is a sequence-independent nucleic acid probe.

[0023] Preferably, a lyoprotectant is also included.

[0024] By adopting the above technical solution and adding a lyophilization protectant, the detection system can be lyophilized to form a loop-mediated isothermal amplification full-component lyophilized ball. After lyophilization, the overall system has a long shelf life and high preservation quality, is easy to store and transport, and can be used in conjunction with a detection device for rapid detection.

[0025] In a third aspect, the present application provides a detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae, which adopts the following technical solution:

[0026] A detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae comprises a first fixing plate and a test strip, wherein the first fixing plate is connected to the test strip.

[0027] Preferably, it also includes a second fixing plate, which is fitted and fixed to the first fixing plate; the first fixing plate is provided with a through test paper hole and a first sample loading hole, a first amplification hole, and a first buffer hole that are interconnected, and the test paper strip is placed in the test paper hole; the second fixing plate is correspondingly provided with a second sample loading hole, a second amplification hole, and a second buffer hole, and the first fixing plate and the second fixing plate are both provided with a through liquid flow hole.

[0028] Preferably, the first amplification well contains freeze-dried beads of all components of loop-mediated isothermal amplification.

[0029] Preferably, it further comprises a third fixing plate, wherein the third fixing plate is provided with corresponding fluid flow holes and chromatography holes which are interconnected.

[0030] In summary, this application has the following beneficial effects:

[0031] 1. This application analyzes the genomes of the two types of Mycoplasma pneumoniae, finds consistent high-copy repeat fragments, and designs 6 primers respectively, which greatly improves the amplification sensitivity. At the same time, a probe detection method is developed to greatly reduce nonspecific amplification and improve detection sensitivity.

[0032] 2. This application also develops a visual integrated detection device, which contains freeze-dried reagents and test strips. Only nucleic acid needs to be added, and the presence or absence of a reaction can be determined by observing the strips after the reaction with the naked eye, without the risk of aerosol contamination.

[0033] 3. The visualization device of the present application has the advantage of real-time detection. The detection of Mycoplasma pneumoniae is convenient, fast and low-cost, and is suitable for rapid screening. The developed fluorescent probe method can be instantly adapted to various fluorescent quantitative detection instruments, which is convenient for inspection units to use quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the design principle of the fluorescent probe and the flow chromatography probe for detecting Mycoplasma pneumoniae by loop-mediated isothermal amplification of the present application.

[0035] Figure 2 It is an overall schematic diagram of the detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae in the present application;

[0036] Figure 3 It is a schematic diagram of an explosion of a detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae in the present application;

[0037] Figure 4 It is a schematic diagram of the test strip interpretation standard for this application.

[0038] 1. First fixed plate; 2. Second fixed plate; 3. Third fixed plate; 4. Test paper hole; 5. Flow hole; 6. Chromatography hole; 7. Sealing strip; 8. Film;

[0039] 11. First sample loading well; 12. First amplification well; 13. First buffer well;

[0040] 21. Second sample loading well; 22. Second amplification well; 23. Second buffer well. DETAILED DESCRIPTION

[0041] The present application is further described in detail below with reference to the accompanying drawings and embodiments.

[0042] A nucleic acid composition for rapid and highly sensitive detection of Mycoplasma pneumoniae comprises an isothermal amplification primer set; the isothermal amplification primer set is used to amplify multiple repeated high-copy fragments in the Mycoplasma pneumoniae genome.

[0043] Specifically, the Tm value of the external primer set should be 55–63°C, and the base length should be 15–25. The Tm value of the 5' end 20 bases of the internal primer set should be higher than the Tm value of other primer sets and controlled at 60–68°C. The Tm value of the 3' end 20 bases of the internal primer set is 55–63°C. The GC content of all primers is controlled at 30%-65%. The stability of the 5' and 3' ends of the primers needs to be ensured, and the formation of dimers between the primers themselves and between primers needs to be avoided. There are strict requirements for the position of the primer anchor on the target sequence. The base length between the external primer sets is controlled at 160-220, and the corresponding bases between the internal primers and the external primers are controlled within 20, and the base length between the internal primer sets is controlled at 120–180. These 4-6 primers can be anchored at 6-8 positions of the nucleic acid, relying on the chain displacement effect of the external primer group and the complementary sequence characteristics of the 5' end of the internal primer group and the template to form an amplification element that can amplify the signal, forming a series of amplification products of different lengths.

[0044] This application designs an isothermal amplification primer set based on the two genotyping genome sequences (U00089 and CP002077) of Mycoplasma pneumoniae M129 and FH in NCBI and analyzes the high copy repeat fragment RepMp5.

[0045] Specifically, the isothermal amplification primer set consists of the following 6 primers:

[0046] The upstream external primer F3 is shown in SEQ ID No. 1;

[0047] Downstream external primer B3 is shown as SEQ ID No.2;

[0048] The upstream internal primer FIP is shown as SEQ ID No. 3;

[0049] The downstream internal primer BIP is shown as SEQ ID No. 4;

[0050] The upstream circularization primer LF is shown in SEQ ID No.5;

[0051] The downstream circularization primer LB is shown as SEQ ID No.6.

[0052] A detection system for rapid and highly sensitive detection of Mycoplasma pneumoniae comprises the above-mentioned nucleic acid composition, a loop-mediated isothermal amplification reaction reagent and an indicator.

[0053] Among them, the prepared nucleic acid composition is specifically as follows: the concentration of the outer primer F3 is 0.3 μM, the concentration of the outer primer B3 is 0.3 μM, the concentration of the inner primer FIP is 1.8 μM, the concentration of the inner primer BIP is 1.8 μM, the concentration of the cyclization primer LF is 1.0 μM, and the concentration of the cyclization primer LB is 1.0 μM.

[0054] The reagents for the loop-mediated isothermal amplification reaction include Bst2.0 DNA polymerase, inorganic pyrophosphatase, dNTP mixture, Tris-HCl, (NH4)2SO4, KCl, 0.1% Tween20 and MgSO4.

[0055] The specific formulation can be Bst2.0 DNA polymerase 8U, inorganic pyrophosphatase 1U, dNTP mixture 1mM, Tris-HCl 20mM, (NH4)2SO4 10mM, KCl 50mM, 0.1% Tween20 and MgSO4 2mM.

[0056] The indicator is a sequence-independent nucleic acid probe, which can be a probe primer set derived from an LF loop primer, a probe primer set derived from an LB loop primer, or a mixture of the two derived probe sets.

[0057] In addition, a freeze-dried protective agent can be added to the detection system to form a loop-mediated isothermal amplification full-component freeze-dried ball. Specifically, the freeze-dried protective agent is trehalose, and the trehalose concentration is best at 10%-20% freeze-dried excipient.

[0058] Therefore, when using different forms of detection systems, you can choose the fluorescent probe method and the flow chromatography probe. And according to different methods, corresponding probe primers are designed as indicators. For the design principle, see Figure 1 .

[0059] Fluorescent probe indicator: The 3' end of the short-chain primer is labeled with the fluorescent group FAM, the concentration is 150-900mM, preferably 300mM, as shown in SEQ ID No. 7; the 5' end of the long-chain primer is labeled with the quenching group BHQ1, the concentration is 150-450mM, preferably 200mM, as shown in SEQ ID No. 8.

[0060] Lateral flow chromatography probe: the 5' end of the short-chain primer is labeled with FAM at a concentration of 150-600 mM, preferably 400 mM, as shown in SEQ ID No.9; the 5' end of the long-chain primer is labeled with biotin at a concentration of 150-400 mM, preferably 300 mM, as shown in SEQ ID No.10.

[0061] Embodiment 1

[0062] The extracted nucleic acid is mixed with the detection system, and the corresponding negative control and positive control are detected by dye isothermal detection. If the detection is effective, the detection is correct, and the result is determined by fluorescence curve analysis. When using the fluorescence detection method, the extracted nucleic acid is mixed with loop-mediated isothermal amplification reaction reagents and fluorescent probes, and can be detected on fluorescent quantitative detection machines such as ABI7500, SLAN-96P, etc. The reaction at 63℃ for 45 minutes can determine the positive and negative of the reaction. If there is a standard S-shaped curve and the Ct value is less than 40, it is positive.

[0063] Embodiment 2

[0064] See also Figure 2 and Figure 3 A detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae comprises a first fixing plate 1, a second fixing plate 2, a third fixing plate 3 and a test strip. The bottom surface of the first fixing plate 1 is fitted and fixed to the top surface of the second fixing plate 2, and the bottom surface of the second fixing plate 2 is fitted and fixed to the top surface of the third fixing plate 3. The top surface of the first fixing plate 1 is sealed by a film 8, and the bottom surface of the third fixing plate 3 is also sealed by a film 8. The test strip is fitted and fixed to the top surface of the first fixing plate 1.

[0065] The first fixed plate 1 is provided with a test paper hole 4 and a first sample loading hole 11, a first amplification hole 12, and a first buffer hole 13 that are interconnected, and the test paper strip is placed in the test paper hole 4; the second fixed plate 2 is correspondingly provided with a second sample loading hole 21, a second amplification hole 22, and a second buffer hole 23, and the first fixed plate 1, the second fixed plate 2, and the third fixed plate 3 are all provided with corresponding through-flow holes 5. The second fixed plate 2 and the third fixed plate 3 are both provided with corresponding through-chromatography holes 6, and the chromatography holes 6 correspond to one end of the test paper strip.

[0066] It should be noted that the second sample loading hole 21, the second amplification hole 22, and the second buffer hole 23 in the second fixed plate 2 are not connected to each other, but are opened at positions corresponding to the first sample loading hole 11, the first amplification hole 12, and the first buffer hole 13, and the first sample loading hole 11 is connected to the second sample loading hole 21, the first amplification hole 12 is connected to the second amplification hole 22, and the first buffer hole 13 is connected to the second buffer hole 23.

[0067] Specifically, the first amplification well 12 and the second amplification well 22 are placed with loop-mediated isothermal amplification full-component freeze-dried balls, the first buffer well 13 and the second buffer well 23 are placed with Tris-HCl buffer with a pH value of 8.0-8.5, and the first sample loading well 11 and the second sample loading well 21 are used to receive nucleic acid samples.

[0068] See also Figure 4 From bottom to top, the test strip consists of a sample pad, a glass fiber pad containing anti-FAM antibodies labeled with colloidal gold, a nitrocellulose membrane labeled with streptavidin and anti-FAM antibodies in sequence, and absorbent paper. The presence or absence of pathogens can be determined by judging the presence or absence of bands on the nitrocellulose membrane.

[0069] Specifically, the nucleic acid to be tested is extracted, added to the first sample loading hole 11 and flows into the second sample loading hole 21, and the first sample loading hole 11 is sealed with the sealing strip 7, and then the device is shaken, at which time the nucleic acid to be tested in the first sample loading hole 11 flows into the first amplification hole 12, and the nucleic acid to be tested in the second sample loading hole 21 flows into the second amplification hole 22, so that the full-component freeze-dried ball of the loop-mediated isothermal amplification is re-dissolved, and the reaction is heated at 63°C for 35 minutes, and then the device is shaken again, so that the buffer solution in the first buffer hole 13 flows into the first amplification hole 12, and at the same time, the buffer solution in the second buffer hole 23 also flows into the first amplification hole 12 along the first buffer hole 13, the reaction liquid and the buffer solution are mixed, and then flow into the chromatography hole 6 in the third fixed plate 3 through the flow hole 5, and then flow into the chromatography hole 6 of the second fixed plate 2 through the chromatography hole 6 of the third fixed plate 3, and further flow into one end of the test strip, and stand for 10 minutes, observe the changes of the C line and T line of the test strip, and judge the positive and negative of the reaction.

[0070] Reference Figure 4If there is a band on the C line, it means that the test process is normal and can bind to the FAM of the short-chain primer of the lateral flow chromatography; if there is a band on the T line, it means that the reaction is positive and can bind to the Biotin of the separated long-chain primer of the lateral flow chromatography.

[0071] Therefore, with the detection device provided in the present application, only nucleic acid needs to be added, and the presence or absence of a reaction can be determined by observing the strips after the reaction with the naked eye, without the risk of aerosol contamination.

[0072] Detection test

[0073] 1. The accuracy and specificity of the isothermal amplification dye method and the isothermal probe method were compared by using the detection rate of clinical samples to analyze the advantages and disadvantages of the methods (see Table 1 for the results). The results of clinical samples were obtained by combining the PCR method and clinical data.

[0074] Table 1 Clinical results of loop-mediated isothermal amplification for detection of Mycoplasma pneumoniae

[0075]

[0076]

[0077] 2. Analyze the nucleic acid extracted from Mycoplasma pneumoniae cultured in standard culture. The detection line of the visual integrated detection device for Mycoplasma pneumoniae was used to quantify the extracted Mycoplasma pneumoniae nucleic acid to 105 copies / μL, and then diluted to 104 copies / μL, 103 copies / μL, 102 copies / μL, 101 copies / μL, and 100 copies / μL in sequence. The detection limits were compared using the fluorescent probe method and the integrated detection device (see the principle and interpretation criteria for details). Figure 1 and Figure 2 ), the results are shown in Table 2.

[0078] Table 2 Detection limits of Mycoplasma pneumoniae detected by the integrated visualization device

[0079]

[0080] Note: + indicates positive, - indicates negative

[0081] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A nucleic acid composition for rapid and highly sensitive detection of Mycoplasma pneumoniae, characterized in that: It comprises an isothermal amplification primer set; the isothermal amplification primer set is used to amplify multiple repeated high-copy fragments in the Mycoplasma pneumoniae genome.

2. The nucleic acid composition for rapid and highly sensitive detection of Mycoplasma pneumoniae according to claim 1, characterized in that: The isothermal amplification primer set consists of the following primers: The upstream external primer F3 is shown in SEQ ID No. 1; Downstream external primer B3 is shown as SEQ ID No.2; The upstream internal primer FIP is shown as SEQ ID No. 3; The downstream internal primer BIP is shown as SEQ ID No. 4; The upstream circularization primer LF is shown in SEQ ID No.5; The downstream circularization primer LB is shown as SEQ ID No.

6.

3. A detection system for rapid and highly sensitive detection of Mycoplasma pneumoniae, characterized in that: The method comprises the nucleic acid composition according to claim 1, a loop-mediated isothermal amplification reaction reagent and an indicator.

4. The detection system for rapid and highly sensitive detection of Mycoplasma pneumoniae according to claim 2, characterized in that: The loop-mediated isothermal amplification reaction reagents include Bst2.0 DNA polymerase, inorganic pyrophosphatase, dNTP mixture, Tris-HCl, (NH4)2SO4, KCl, 0.1% Tween20 and MgSO4.

5. The detection system for rapid and highly sensitive detection of Mycoplasma pneumoniae according to claim 2, characterized in that: The indicator is a sequence-independent nucleic acid probe.

6. The detection system for rapid and highly sensitive detection of Mycoplasma pneumoniae according to claim 2, characterized in that: Lyoprotectants are also included.

7. A detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae, characterized in that: It comprises a first fixing plate (1) and a test strip, wherein the first fixing plate (1) is connected to the test strip.

8. A detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae according to claim 6, characterized in that: it also includes a second fixing plate (2), the second fixing plate (2) is fixed to the first fixing plate (1); the first fixing plate (1) is provided with a through test paper hole (4) and a first sample addition hole (11), a first amplification hole (12), and a first buffer hole (13) that are interconnected, and the test paper strip is placed in the test paper hole (4); the second fixing plate (2) is correspondingly provided with a second sample addition hole (21), a second amplification hole (22), and a second buffer hole (23), and the first fixing plate (1) and the second fixing plate (2) are both provided with a through flow hole (5).

9. The detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae according to claim 7, characterized in that: The first amplification well (12) contains a freeze-dried ball of all components of loop-mediated isothermal amplification.

10. The detection device for rapid and highly sensitive detection of Mycoplasma pneumoniae according to claim 6, characterized in that: It also comprises a third fixing plate (3), wherein the third fixing plate (3) is provided with corresponding liquid flow holes (5) and chromatography holes (6) which are interconnected.

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