Primers, crRNA and their applications for broad-spectrum detection of mycoplasma based on LAMP-CRISPR / Cas12a

Abstract

The present invention discloses a primer, crRNA and application thereof for broad-spectrum detection of mycoplasma based on LAMP-CRISPR / Cas12a, and relates to the technical field of mycoplasma detection. The primer and crRNA combination provided by the present invention includes 2 outer primers, 5 inner primers, 4 loop primers and crRNA. Based on this, the present invention also provides a LAMP-CRISPR / Cas12a system and product for broad-spectrum detection of mycoplasma, as well as a method for detecting mycoplasma that is not for the purpose of disease diagnosis and treatment. The primer and crRNA combination provided by the present invention and the corresponding detection method can simultaneously meet the requirements of specificity, specificity, detection coverage (broad-spectrum detection) and sensitivity stipulated by various countries and the European Pharmacopoeia; the operation is simple, the cost is low, the time consumption is short, the detection efficiency is high, the dependence on expensive instruments is low, and the instant detection of mycoplasma can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of mycoplasma detection, and in particular to a mycoplasma broad-spectrum detection primer combination based on LAMP-CRISPR / Cas12a. Background Art

[0002] Mycoplasma belongs to the class of Soft-coated Fungi. Its cells are soft, simple in structure, and polymorphic. They are 0.3-0.8μm in diameter and can pass through the commonly used sterilization filter membrane with a diameter of 0.45μm. They are the smallest self-replicating microorganisms discovered so far. In 2002, Drexler and other scholars conducted statistics on mycoplasma contamination in 440 lymphoma cell lines tested, and only 64% of the cell lines were free of mycoplasma contamination. Survey data from German DSMZ cell bank researchers showed that about 1 / 4 of the cells were affected by mycoplasma contamination. Mycoplasma contamination can have a great impact on scientific research or the production of biological products. For example, it can hinder cell metabolism, change cell morphology, change cell signaling pathways, inhibit hybridoma cell fusion, and replace monoclonal antibodies with anti-mycoplasma antibodies. These effects are often fatal in the production of biological products.

[0003] In view of the potential impact of mycoplasma contamination on the stability and safety of biological products, the corresponding regulatory authorities of various countries have also made certain regulations on the control of mycoplasma contamination in the production process of biological products. The Mycoplasma Detection Method in Chinese Pharmacopoeia 3301 points out that when the master cell bank, working cell bank, virus seed batch, control cell and clinical treatment cell are tested for mycoplasma, the culture method and indicator cell culture method (DNA staining method) should be carried out at the same time. The virus harvest fluid and stock solution of viral vaccines are tested for mycoplasma by culture method; if necessary, the indicator cell culture method can also be used to screen the culture medium. Other methods approved by the national drug testing agency can also be used. Chapter 2.6.2 of the European Pharmacopoeia, General Chapter 63 of the United States Pharmacopoeia and Chapter G3 of the Japanese Pharmacopoeia also stipulate the control of mycoplasma contamination of biological products.

[0004] With the continuous enrichment of biological products, such as drugs based on gene therapy, cell therapy and tissue engineering, these drugs have a short shelf life and are difficult to store, requiring a shorter detection cycle. For this reason, many national pharmacopoeias have introduced nucleic acid amplification techniques (NAT). For example, the European Pharmacopoeia points out that NAT technology or other properly validated technologies can replace culture methods or indicator cell methods, and points out that A.laidlawii, M.fermentans, M.hyorhinis, M.orale, M.pneumoniae, M.gallisepticum, M.synoviae, M.arginini and Spiroplasma citri are used for detection line validation, and Clostridium, Lactobacillus and Streptococcus are used for specificity validation. The Japanese Pharmacopoeia points out that when testing mammalian cells, A.laidlawii, M.arginini, M.fermentans, M.hyorhinis, M.orale, M.pneumoniae, and M.salivarium should be used for verification. When testing insect cells or plant cells, S.citri should also be verified. When testing avian cells, M.synoviae should also be verified. It also points out that the sensitivity of NAT detection should reach 100CFU / ml when replacing the indicator cell method for detecting mycoplasma. The sensitivity of NAT detection should reach 10CFU / ml when replacing the culture method for detecting mycoplasma. The United States Pharmacopoeia also indicates that NAT technology can replace the culture method and indicator cell method for the detection of mycoplasma.

[0005] Loopmediated isothermal amplification (LAMP) is a common nucleic acid amplification technology that does not require thermal cycling, denaturation, annealing, and extension stages, and can obtain a large amount of target products in a short time. However, LAMP technology often requires the participation of 4-6 primers during amplification, and nonspecific amplification is prone to occur. The probability of false positives is high when using LAMP amplification technology alone for detection. LAMP amplification technology has extremely high requirements for amplification length. The sensitivity of detection using LAMP amplification technology alone is low, and its nonspecific amplification will also affect the detection sensitivity. When detecting multiple mycoplasmas, the difficulty of primer design increases exponentially with the increase in the number of detection objects, and the possibility of mutual interference between primers will also greatly increase. Even if professional software-assisted design is used, the above problems cannot be solved and the effect is extremely poor. These problems limit its application in broad-spectrum instant detection of mycoplasmas.

[0006] The CRISPR-Cas12 (Clustered Regularly Interspaced Short PalindromicRepeats, CRISPRs) system is a self-defense system evolved in the struggle between bacteria and viruses. When the specific cleavage activity of the CRISPR / Cas12 protein family is activated, its accompanying nonspecific cleavage activity is also activated. It has extremely strong recognition specificity and extremely high target cleavage efficiency. Therefore, in addition to being used for gene editing, it has also been widely used in in vitro diagnosis.

[0007] In the prior art, Chinese patent application CN105755161A discloses a LAMP primer set for broad-spectrum detection of mycoplasma, including 2 outer primers, 2 inner primers and 2 loop primers. The patent uses the ITS-23srRNA sequences of 8 common mycoplasmas in cell culture, M.hyorhinis, M.fermentans, M.arginini, M.hominis, M.orale, M.salivarium, M.pirum, and AcholeplasmaLaidlawii, to find specific conserved target gene fragments (23s rRNA sequence 5' end region) through homologous sequence analysis and comparison, and uses primerexplorerV4 online design to obtain a LAMP primer combination consisting of 2 outer primers, 2 inner primers and 2 loop primers. After analysis and comparison, although the patent application lists multiple mycoplasmas, the patent selects the conserved regions of some mycoplasma sequences and does not fully include the verification of several mycoplasmas specified in the pharmacopoeia. These mycoplasmas are closely related and relatively conservative in sequence, so the detection results are prone to mutual interference. This patent application only performs LAMP amplification without combining it with other technologies, so the sensitivity and coverage are low, and the sensitivity does not meet the requirements of various pharmacopoeias.

[0008] The document "Establishment and Application of a New Method for Detecting Mycoplasma Pneumoniae" (Zhou Lijuan et al., Journal of Practical Clinical Medicine, Issue 19, 2022, pp. 100-104) discloses the rapid detection of Mycoplasma pneumoniae by combining CRISPR-Cas12a technology with LAMP single-tube one-step method, using Primer Explorer Version 5 software to design LAMP primers and Deskgen software to design CRISPR / Cas12a crRNA. The sample source of this document is clinical samples, and it is impossible to detect a wider range of samples. This document only designed 4 primers, lacked loop primers, and had poor sensitivity.

[0009] Chinese patent application CN114410753A discloses a LAMP primer combination for detecting mycoplasma obtained by aligning the common conservative sequences of 16S rRNA sequences of Mycoplasma hyorhinis, Mycoplasma fermentans, Mycoplasma arginine, Mycoplasma hominis, Mycoplasma oralis, Mycoplasma salivarius, Mycoplasma pear, Mycoplasma acholereticum, Mycoplasma agalactiae, Mycoplasma bovis, Mycoplasma maxillaryis, Mycoplasma arthritis and Mycoplasma pulmonary using snapgene software. The F / B primer used in the patent was found to have no amplification primers through primer-blast search on the NCBI website. In theory, it is difficult to achieve amplification using PCR technology, which is extremely difficult for LAMP amplification technology; the patent application only uses one loop primer, which is theoretically difficult to achieve high-sensitivity detection of multiple mycoplasmas.

[0010] Currently, there is no instant technology that combines LAMP and CRISPR-Cas12 for the detection of multiple mycoplasmas. Summary of the invention

[0011] In response to the problems and technical gaps in the existing technology, this patent has carried out a complete specificity verification. It not only tests several bacteria that need to be verified as specified in the European Pharmacopoeia, but also verifies the specificity of common contaminating bacteria, fungi and cell lines commonly used in biological products. This is not supported by data in other patent documents, especially the verification of several closely related bacterial species specified in the Pharmacopoeia. This patent adds an amplification link based on Cas nucleic acid detection technology on the basis of LAMP amplification technology, further increasing the sensitivity of mycoplasma detection. While meeting the sensitivity and specificity requirements specified in the Pharmacopoeia, it can cover the instant detection of multiple mycoplasmas.

[0012] The present invention compares the 16s rRNA-23s rRNA regions of a variety of common mycoplasmas and bacteria, selects appropriate regions to design and obtain LAMP amplification primer combinations based on the conservation between mycoplasmas and the differences with other bacteria, and finally retains 2 outer primers, 5 inner primers, and 4 loop primers through screening and optimization. The present invention also selects specific regions of 2 mycoplasma gene sequences to design crRNA targets, and uses CRISPR / Cas12a enzymes to design and screen the designed crRNAs, and finally retains 4 crRNAs. Finally, the present invention uses it as a primer and crRNA combination for broad-spectrum detection of mycoplasmas. The combination can simultaneously meet the requirements of specificity, specificity, detection coverage (broad-spectrum detection) and sensitivity of mycoplasma detection. The detection capability of this patent is predicted by NCBI-blast comparison. In theory, it can effectively detect at least 112 mycoplasmas in a broad spectrum, and there is no cross-reaction with bacteria, fungi, biological products, and commonly used cell lines for scientific research. It is specifically achieved by the following technologies.

[0013] The present invention provides a combination of mycoplasma broad-spectrum detection primers and crRNA based on LAMP-CRISPR / Cas12a, comprising 2 outer primers, 5 inner primers, 4 loop primers and crRNA, the nucleotide sequences of the outer primers are shown in SEQ ID NOs.1 and 2, the nucleotide sequences of the inner primers are shown in SEQ ID NOs.3-7, and the nucleotide sequences of the loop primers are shown in SEQ ID NOs.8-11; the nucleotide sequences of the crRNA are shown in SEQ ID NOs.12-15, respectively.

[0014] Furthermore, the primer pairs for preparing the crRNA are F1 / R1, F2 / R2, F3 / R3 and F4 / R4, and the nucleotide sequences of the primer pairs F1 / R1, F2 / R2, F3 / R3 and F4 / R4 are shown in SEQ ID NO.16-23, respectively.

[0015] The present invention also provides a LAMP-CRISPR / Cas12a system for detecting mycoplasma, wherein the LAMP-CRISPR / Cas12a system comprises the nucleotide sequences of the outer primers shown in SEQ ID NOs.1 and 2, the inner primers shown in SEQ ID NOs.3-7, and the loop primers shown in SEQ ID NOs.8-11;

[0016] The LAMP-CRISPR / Cas12a system also includes the crRNA shown in SEQ ID NO.12-15; or, it also includes primer pairs F1 / R1, F2 / R2, F3 / R3 and F4 / R4, and the nucleotide sequences of the primer pairs F1 / R1, F2 / R2, F3 / R3 and F4 / R4 are shown in SEQ ID NO.16-23, respectively.

[0017] Furthermore, the LAMP-CRISPR / Cas12a system also includes Cas12a protein, single-stranded DNA signal molecule, 10× buffer, RNasin and nuclease-free water.

[0018] The present invention also provides a product for detecting mycoplasma, which comprises any one of the above-mentioned LAMP-CRISPR / Cas12a systems provided by the present invention.

[0019] Furthermore, the product is a detection kit, a detection test strip or a detection chip.

[0020] The present invention also provides a method for detecting mycoplasma that is not intended for disease diagnosis and treatment, the steps of which are: pretreating a test sample, extracting the genome of the test sample, amplifying the genome using the genome as a template, using the amplified product as a template, using any one of the above-mentioned LAMP-CRISPR / Cas12a systems provided by the present invention, or using any one of the above-mentioned products provided by the present invention for detection and result determination.

[0021] It should be noted that the LAMP-CRISPR / Cas12a system and product for detecting mycoplasma provided by the present invention are used for non-disease diagnosis and treatment purposes. For example, it can be used to quickly detect mycoplasma contamination in air, water, soil, article instrument surfaces, waste liquid, laboratories and other environments, and can also be used to detect mycoplasma contamination in biological products. That is, all situations that are not directly used to diagnose or treat diseases or symptoms related to mycoplasma in humans or animals can use the technical means of the present invention. Of course, in order to diagnose or treat diseases or symptoms related to mycoplasma in humans or animals, the technical means of the present invention can also be used.

[0022] Furthermore, in the above method, the detection method is fluorescence method or lateral flow method.

[0023] Furthermore, the method for determining the result is:

[0024] When the fluorescence method is used, if the test sample has an obvious fluorescence growth curve and the fluorescence value is greater than 15 cfu, the test sample can be determined to be mycoplasma positive, otherwise it is negative;

[0025] When the lateral flow method is used, if there is a bright band in the detection line reading area of ​​the test strip at 5 minutes, the test sample can be judged as mycoplasma positive, otherwise it is negative.

[0026] Compared with the prior art, the present invention is beneficial in that:

[0027] 1. The present invention combines LAMP isothermal amplification technology, CRISPR / Cas12a technology, and fluorescence / lateral flow detection technology to provide a primer and crRNA combination for broad-spectrum detection of mycoplasma. After rapid amplification of mycoplasma nucleic acid (test sample), the combination can accurately identify the amplification product and finally present the results using fluorescence method or simple lateral flow technology.

[0028] 2. The primer and crRNA combination provided by the present invention can simultaneously meet the requirements of specificity, specificity, detection coverage (broad-spectrum detection) and sensitivity for mycoplasma detection. According to the high similarity comparison results of the NCBI biological resource database, the combination can effectively detect 112 types of mycoplasmas; it can achieve the detection of 10 copies / reaction system or 5 cfu / ml concentration of 10 types of mycoplasmas clearly indicated in the European Pharmacopoeia and the Japanese Pharmacopoeia.

[0029] 3. Compared with the culture method and indicator cell method used in the Chinese Pharmacopoeia, the primer and crRNA combination provided by the present invention and the corresponding method for detecting mycoplasma have the advantages of simple operation, short time consumption and high sensitivity. The detection cycle of the culture method in the Chinese Pharmacopoeia is at least 14 days, and the detection of mycoplasma species is easily interfered by the components of the culture medium. The detection cycle of the indicator cell method in the Chinese Pharmacopoeia is 3-5 days. The primer and crRNA combination and the corresponding method provided by the present invention have higher amplification efficiency than the fluorescence quantitative method, and can complete mycoplasma detection as low as 5CFU / ml concentration within 1 hour (LAMP amplification time is about 50min) after DNA extraction.

[0030] 5. The primer and crRNA combination provided by the present invention and the corresponding method for detecting mycoplasma simultaneously use LAMP amplification technology and CRISPR / Cas12a detection technology, which is equivalent to further confirming the LAMP amplification product and further amplifying the detection signal, that is, two signal amplifications and confirmations. Compared with simple LAMP or ordinary PCR technology, the method of the present invention has stronger specificity and higher sensitivity.

[0031] 6. Compared with other LAMP and CRISPR / Cas combined technologies, the method provided by the present invention optimizes the primer design idea of ​​LAMP amplification of mycoplasma, can use shorter conserved regions to amplify more mycoplasmas, and improve the reaction efficiency of LAMP technology (LAMP technology has extremely high requirements for the length of the amplified fragment). Combining CRISPR / Cas technology can make up for the poor specificity of LAMP technology in amplifying mycoplasma in this method, and ultimately achieve higher coverage and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of Example 3 in which the FC and BC regions are partially overlapped when designing LAMP amplification primers to make full use of the conservative regions; the figure shows the F1 region and the B1 region;

[0033] Figure 2 This is a schematic diagram of designing BIP primers for multiple regions to improve amplification efficiency when designing LAMP amplification primers in Example 3; the figure shows multiple B2 regions;

[0034] Figure 3 The results of using bacteria and fungi to verify the specificity of the detection system of the present invention by fluorescence method within 40 minutes. In the figure, 1 is a positive control sample, 2-12 are Lactobacillus acidophilus (L.acidophilus), Streptococcus pneumoniae (S.pneumoniae), Clostridium acetobutylicum (C.acetobutylicum), Escherichia coli (E.coli), Bacillus subtilis (B.subtilis), Pseudomonas aeruginosa (P.aeruginosa), Clostridium sporogenes (C.sporogenes), Mycobacterium phleum (M.phle), Staphylococcus aureus (S.aureus), Aspergillus niger (A.niger) samples and NTC samples respectively;

[0035] Figure 4 The results of using bacteria and fungi to verify the specificity of the detection system of the present invention by fluorescence method at 40 minutes. Figure 1 same;

[0036] Figure 5 The results within 40 minutes of fluorescence verification of the detection specificity of the detection system of the present invention using cells commonly used in the production of biological products. In the figure, 1 is a positive control sample, and 2-11 are CHO-K1, MSC, A9, Walker-256, vero, MDBK, PK-15, Hi-5, sf9 cell samples and NTC samples respectively;

[0037] Figure 6 The results of the fluorescence method verification of the detection specificity of the detection system of the present invention using cells commonly used in the production of biological products at 40 minutes. Figure 1 same;

[0038] Figure 7-16 They are respectively the sensitivity verification results of the present invention. Specifically, Figure 7-16 The fluorescence changes within 40 minutes of the detected oral mycoplasma (M.orale), hyorhinis, synoviae, fermentans, arginini, pneumonia, A.laidlawii, S.citri, gallisepticum and salivarium are shown in Figure 1. In each figure, 1-7 represent 5 copies / reaction, 7 copies / reaction, 10 copies / reaction, 100 copies / reaction, 1000 copies / reaction, 10000 copies / reaction and NTC sample results, respectively;

[0039] Fig.17 The results are the results of the verification of the alternativeness of the pharmacopoeia method. Specifically, they are the fluorescence kinetic detection results within 40 minutes of 5 CFU / ml concentration of oral mycoplasma (M.orale), hyorhinis, synoviae, fermentans, arginini, pneumoniae, A.laidlawii, S.citri, gallisepticum, salivarium and negative control samples;

[0040] Fig.18 The results are the results of the verification of the alternativeness of the pharmacopoeia method. Specifically, they are the fluorescence kinetic detection results of 5 CFU / ml concentration of oral mycoplasma (M.orale), hyorhinis mycoplasma (M.hyorhinis), synoviae mycoplasma (M.synoviae), fermentans mycoplasma (M.fermentans), arginini mycoplasma (M.arginini), pneumonia mycoplasma (M.pneumoniae), A.laidlawii, S.citri, M.gallisepticum and M.salivarium and negative control samples at 40 minutes;

[0041] Fig.19 The results of mycoplasma detection using lateral flow technology in the present invention. 1-11 are the strip banding results of oral mycoplasma (M.orale), hyorhinis mycoplasma (M.hyorhinis), synovial fluid mycoplasma (M.synoviae), fermentation mycoplasma (M.fermentans), arginine mycoplasma (M.arginini), pneumonia mycoplasma (M.pneumoniae), laidlawii, citrioplasma (S.citri), gallisepticum mycoplasma (M.gallisepticum) and salivarium (M.salivarium) at a concentration of 5 CFU / ml and the negative control sample at 5 minutes;

[0042] Fig. 20 and 21 The sensitivity verification results of oral mycoplasma detection when the conventional primer design scheme was used to design the detection primers and crRNA combination based on LAMP-CRISPR / Cas12a. Fig. 20 and21 1-7 represent 5 copies / reaction, 7 copies / reaction, 10 copies / reaction, 100 copies / reaction, 1000 copies / reaction, 10000 copies / reaction and NTC sample results, respectively.

[0043] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The terms provided in the present invention all adopt definitions that are easily known or well-known to those skilled in the art.

[0045] (1) Detection

[0046] Detection means to test or examine a substance or material for the presence or absence of a substance or material, such as, but not limited to, a chemical substance, an organic compound, an inorganic compound, a metabolite, a drug or a drug metabolite, an organic tissue or a metabolite of an organic tissue, a nucleic acid, a protein, or a polymer. In addition, detection means to test the amount of a substance or material. Furthermore, assay also means immunoassay, chemical assay, enzyme assay, etc.

[0047] (2) Sample (i.e. test article)

[0048] Samples that can be detected by the detection reagent or method of the present invention include biological fluids (e.g., case fluids or clinical samples). Liquid samples or fluid samples can be derived from solid or semi-solid samples, including excreta, biological tissues, and food samples. Solid or semi-solid samples can be converted into liquid samples by any appropriate method, such as mixing, mashing, macerating, incubating, dissolving, or digesting solid samples by enzymolysis in a suitable solution (e.g., water, PBS or other buffer).

[0049] "Biological samples" include samples from animals, plants and food, such as urine, saliva, blood and its components, cerebrospinal fluid, vaginal secretions, sperm, feces, sweat, secretions, tissues, organs, tumors, tissue and organ cultures, cell cultures and media from humans or animals. Preferably, the biological sample is urine or a swab. Food samples include food processing materials, final products, meat, cheese, wine, milk and drinking water.

[0050] "Plant sample" includes any plant, plant tissue, plant cell culture and media.

[0051] "Environmental samples" are derived from the environment (eg, liquid samples from lakes or other water bodies, sewage samples, soil samples, groundwater, seawater and waste liquid samples). Using the present invention and appropriate detection elements, any analyte can be detected.

[0052] The reagents and instruments used in the following specific embodiments, unless otherwise specified, can all be purchased through conventional channels. The reagents or reaction raw materials used, unless otherwise specified, all use the initial concentration of each raw material in the purchased goods.

[0053] Example 1: Design and preparation of crRNA.

[0054] 1. Design of crRNA for CRISPR / Cas12a detection technology

[0055] By comparing the 16s rRNA and 23s rRNA genes of various common mycoplasmas and other Gram-positive bacteria (pharmacopoeia indications), crRNA was designed in the region specific to the mycoplasma gene sequence based on the conservation among mycoplasmas and the differences between mycoplasmas and other Gram-positive bacteria. Four crRNAs with the best specificity and sensitivity were obtained through screening. The nucleotide sequences of these four crRNAs are shown in SEQ ID NO.12-15.

[0056] At the same time, four pairs of primers F1 / R1, F2 / R2, F3 / R3 and F4 / R4 were designed for synthesizing these four crRNAs, and their nucleotide sequences are shown in SEQ ID NOs.16-23, respectively.

[0057] 2. Preparation of crRNA

[0058] (1) In vitro transcription template synthesis using DNA annealing buffer (Solabo, D2810)

[0059] Prepare the reaction system as shown in Table 1 below.

[0060] Table 1

[0061]

[0062] Place the prepared reaction solution in a PCR instrument, heat the sample to 95°C, and cool the sample to 25°C at a cooling rate of 0.1°C / s; finally, store the PCR product at -20°C, or proceed directly to subsequent operations.

[0063] (2) Use DNA recovery kit (NEB, D4033) to recover in vitro transcription template

[0064] Add 5 volumes of DNA binding buffer to the PCR product, mix the mixture, add the above solution to the Zymo-Spin™ Column2 collection column, and centrifuge at 8000g for 30s. Add 200μl of DNA wash buffer and centrifuge at 9000g for 30s (repeat once). Discard the filtrate, put the column back into the centrifuge, and centrifuge at 12000g for 3min. Add 20μl RNA-free water to the collection column, incubate at room temperature for 2min, and centrifuge at 9000g for 2min to collect the template.

[0065] (3) Perform in vitro transcription using an in vitro transcription kit (NEB, E2050S).

[0066] Prepare the reaction system as shown in Table 2 below.

[0067] Table 2

[0068]

[0069] After mixing evenly, centrifuge briefly, incubate at 37°C for 5 h, add 45 μl of nuclease-free water, then add 2 μl of DNase I, mix well and incubate at 37°C for 20 min.

[0070] (4) Recover crRNA using RNA recovery kit (NEB, T2040L)

[0071] Add 2 volumes of RNA Cleanup Binding Buffer to the transcription product and mix the mixture; add an equal volume of anhydrous ethanol to the above solution, mix gently (no vortexing is required), and let stand for 1-2 minutes; add the above solution to the collection column, centrifuge at 8000g for 1 minute, discard the filtrate, add 500μl of RNA Cleanup Wash Buffer, centrifuge at 9000g for 1 minute (repeat once), discard the filtrate, put the column back into the centrifuge, and centrifuge at 12000g for 3 minutes; add 50μl RNA-free water to the collection column, centrifuge at 9000g for 2 minutes to collect crRNA.

[0072] Example 2: Preparation of samples (test sample, positive control sample and negative control sample)

[0073] 1. Sample pretreatment

[0074] (1) Pretreatment of test samples:

[0075] If the test sample is a cell suspension: After centrifugation at 500g for 5 minutes, take 1 ml of the supernatant into a 1.5 ml centrifuge tube, centrifuge at 16000g for 10 minutes, discard 0.8 ml of the supernatant, and store the remaining 0.2 ml of the solution at -80℃ or proceed directly to the next step.

[0076] If the test sample (without cells) is a non-cellular biological preparation: directly take 0.2 ml of the test sample into a 1.5 ml centrifuge tube and store it at -80°C or proceed directly to the next step.

[0077] (2) Pretreatment of positive control samples

[0078] A mixture of 200 copies of the genome of Mycoplasma oralis and 200 copies of the genome of Mycoplasma pneumoniae was added to the test sample; then the same treatment as the test sample was used.

[0079] (3) Pretreatment of negative control samples

[0080] The negative sample was 1 ml of MEM basal medium containing 5% FBS, and the pretreatment method was the same as the test sample.

[0081] 2. Genome extraction

[0082] Before extraction, prepare crRNA solution and protease solution according to the instructions of the genome extraction kit (Qiagen, 57704, the kit includes crRNA), and then use the genome extraction kit (Qiagen, 57704) to extract the DNA of the sample according to the following operation:

[0083] (1) Add 25 μl of protease solution to 200 μl of sample to be extracted (if the sample volume is less than 200 μl, use 0.9% sodium chloride solution to make up), then add 200 μl of buffer AL and gently shake using a vortexer for 15 seconds;

[0084] (2) Place in a 56°C water bath, remove the sample tube after 15 minutes and allow it to cool to room temperature, then add 250 μl of anhydrous ethanol. If the sample temperature exceeds 25°C, ethanol must be precooled before addition, and the sample is gently shaken on a vortex for 15 seconds, then left at room temperature for 5 minutes;

[0085] (3) Add the treated sample to the adsorption column and centrifuge at 6000g for 1 min;

[0086] (4) Discard the collection tube containing the waste liquid, place the adsorption column in a new collection tube, add 500 μl of anhydrous ethanol to the collection column, and centrifuge at 6000 g for 1 min;

[0087] (5) Discard the collection tube containing the waste liquid and place the adsorption column in a new collection tube; add 500 μl of AW1 and centrifuge at 6000 g for 1 min;

[0088] (6) Discard the collection tube containing the waste liquid, place the adsorption column in a new collection tube and add 500 μl of AW2, and centrifuge at room temperature for 1 min;

[0089] (7) Place the adsorption column into a new collection tube, open the cover of the adsorption column, and place it at 56°C for 3 minutes.

[0090] (8) Place the adsorption column in a new 1.5 ml EP tube and add 80 μl of molecular-grade water to the center of the adsorption column. After leaving it at room temperature for 5 min, centrifuge to collect the DNA solution and proceed directly to subsequent operations or store it at -20 °C for later use.

[0091] Example 3: LAMP amplification

[0092] 1. Screening and optimization of LAMP amplification primers

[0093] The present invention partially overlaps the FC and BC regions, fully utilizes the conservative region, and designs multiple BIPs to improve the amplification efficiency, such as Figure 1 , 2 Finally, two outer primers, five inner primers and four loop primers were obtained by screening and optimization. The nucleotide sequences of the two outer primers are shown in SEQ ID NOs. 1 and 2, the nucleotide sequences of the five inner primers are shown in SEQ ID NOs. 3-7, and the nucleotide sequences of the four loop primers are shown in SEQ ID NOs. 8-11.

[0094] The specific sequence is shown in Table 3 below.

[0095] Table 3

[0096]

[0097]

[0098] 2. LAMP amplification

[0099] According to the sequences in Table 3 above, prepare the primer mixtures LAMP-MY-16s-F, LAMP-MY-16s-B, LAMP-MY-16s-LoopF1 / 2 / 3, LAMP-MY-16s-LoopB, LAMP-MY-16s-FIP1 / 2 and LAMP-MY-16s-BIP1 / 2 / 3.

[0100] Prepare the reaction system shown in Table 4 on ice:

[0101] Table 4

[0102]

[0103] Place the prepared reaction system in a 65°C water bath or metal thermostat, react for 50 minutes, and then proceed directly to the subsequent operation or store at -80°C for future use.

[0104] Example 4: Detection and result determination based on CRISPR-Cas12a nucleic acid detection system

[0105] 1. Fluorescence detection process

[0106] The fluorescence detection system is shown in Table 5 below.

[0107] Table 5

[0108]

[0109] Fluorescence detection was performed using a multifunctional microplate reader (Thermo Fisher, Varioskan LUX) in the kinetic detection mode, at 38°C, with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0110] 2. Fluorescence test results

[0111] The method for determining the results of the fluorescence method is: the positive control has an obvious fluorescence growth curve, the negative control has no obvious fluorescence change, and the fluorescence value of the negative control is below 15 cfu. If the test sample has an obvious fluorescence growth curve and its fluorescence value is greater than 15 cfu, the test sample can be determined to be mycoplasma positive, otherwise it is negative.

[0112] (1) Specificity verification

[0113] Using the DNA nucleic acid purchased from MB Company as a template, specificity verification was performed according to the method of Experimental Example 3.

[0114] like Figure 3 and 4 As shown, Figure 3 and 4 The results are within 40 minutes and at 40 minutes respectively, when the specificity of the detection system of the present invention is verified by fluorescence method using bacteria and fungi. Figure 3 and 4Among them, 1 is the positive control sample, and 2-12 are Lactobacillus acidophilus (L.acidophilus), Streptococcus pneumoniae (S.pneumoniae), Clostridium acetobutylicum (C.acetobutylicum), Escherichia coli (E.coli), Bacillus subtilis (B.subtilis), Pseudomonas aeruginosa (P.aeruginosa), Clostridium sporogenes (C.sporogenes), Mycobacterium phle (M.phle), Staphylococcus aureus (S.aureus), Aspergillus niger (A.niger) samples and NTC samples, respectively.

[0115] from Figure 3 and 4 It can be seen that the fluorescence intensity curve of the positive control sample No. 1 rises rapidly, reaching a maximum value of more than 400 at about 12 minutes, and then remains basically unchanged until 40 minutes; while the fluorescence intensity curves of No. 2-12 are basically overlapped within 0-40 minutes, and are basically horizontal straight lines, and the fluorescence intensity is always close to 0. This shows that the detection results of the mycoplasma positive control using the crRNA of the present invention have significant specificity relative to common bacteria and fungi.

[0116] like Figure 5 and 6 As shown, Figure 5 and 6 The results are respectively the results within 40 minutes and at 40 minutes of fluorescence verification of the detection specificity of the detection system of the present invention using cells commonly used in the production of biological products. Figure 5 and 6 In the figure, 1 is the positive control sample, and 2-11 are CHO-K1, MSC, A9, Walker-256, vero, MDBK, PK-15, Hi-5, sf9 cell samples and NTC samples, respectively.

[0117] from Figure 5 and 6 It can be seen that the fluorescence intensity curve of the positive control No. 1 rises rapidly, reaching a maximum value of more than 400 at about 12 minutes, and then remains basically unchanged until 40 minutes; while the fluorescence intensity curves of No. 2-12 completely overlap within 0-40 minutes, and are basically horizontal lines, and the fluorescence intensity always approaches 0. This shows that the detection results of the mycoplasma positive control using the crRNA of the present invention have significant specificity relative to the common 9 cell lines.

[0118] (2) Sensitivity verification

[0119] Using the DNA nucleic acid purchased from MB Company (kit) as a template, sensitivity verification was performed according to the method of Experimental Example 3.

[0120] like Figure 7-16 As shown, Figure 7-16 The fluorescence changes within 40 minutes of the detected oral mycoplasma (M.orale), hyorhinis, synoviae, fermentans, arginini, pneumonia, A.laidlawii, S.citri, gallisepticum and salivarium are shown in Figure 1. In each figure, 1-7 represent 5 copies / reaction, 7 copies / reaction, 10 copies / reaction, 100 copies / reaction, 1000 copies / reaction, 10000 copies / reaction and NTC sample results, respectively.

[0121] from Figure 7-16 It can be seen that within the range of 0-40min, the fluorescence intensity of 10 mycoplasmas with 100 copies / reaction all rose rapidly, and the final fluorescence intensity reached more than 400. This shows that the crRNA provided by the present invention also has a very high sensitivity to mycoplasmas with 10 copies / reaction. It also has a very high sensitivity to some mycoplasmas with 5 copies / reaction (such as Mycoplasma hyorhinis, Acholeraplasma leishmanii and Spiroplasma citrinum), or 7 copies / reaction (such as Mycoplasma synovium, Mycoplasma fermentum, Mycoplasma pneumoniae and Mycoplasma salivarius), or 10 copies / reaction (such as Mycoplasma oralis and Mycoplasma gallisepticum).

[0122] (3) Broad-spectrum verification

[0123] Based on the verification results of the specificity and sensitivity of the above-mentioned patent system for the detection of mycoplasmas indicated in the pharmacopoeias of various countries, and the fact that the mycoplasmas indicated in the pharmacopoeias are widely distributed and have large sequence differences, it also indirectly reflects that the combination of mycoplasma broad-spectrum detection primers and crRNA based on LAMP-CRISPR / Cas12a of the present invention can achieve broad-spectrum detection of multiple mycoplasmas.

[0124] (4) Verification of the interchangeability of pharmacopoeial methods

[0125] A standard mycoplasma strain was selected and the method of Examples 2 and 3 was used for verification.

[0126] like Fig.17 and 18 As shown, Fig.171-11 are the fluorescence kinetic detection results within 40 min of oral mycoplasma (M.orale), hyorhinis, synoviae, fermentans, arginini, pneumoniae, A.laidlawii, S.citri, M.gallisepticum and M.salivarium at a concentration of 5 CFU / ml and the negative control sample. Fig.18 The corresponding results are the fluorescence kinetic detection results of the above-mentioned mycoplasma and negative control samples at 40 minutes.

[0127] from Fig.17 and 18 It can be seen that compared with the negative control sample, the crRNA provided by the present invention can significantly increase the fluorescence intensity within 25 minutes for 10 common mycoplasmas at a concentration of 5 CFU / mL (the minimum detection line specified in the pharmacopoeia is 10 CFU / ml) and reach a maximum value of more than 400, and the fluorescence intensity at 5 minutes is also significantly higher than the negative control. This shows that the method of the present invention shows very significant specificity and sensitivity for mycoplasma detection, and can complete the alternative verification of the pharmacopoeia method according to the requirements of the pharmacopoeia.

[0128] 3. Lateral flow technology detection method

[0129] The lateral flow technology detection system is shown in Table 6 below.

[0130] Table 6

[0131]

[0132] The above reaction system was placed in a 38°C constant temperature box for reaction. After 10 minutes, the Cas12 special nucleic acid detection test strip (Bolex, M20801-F007) was placed in the above reaction system. After 3 minutes, the results were observed and photographed.

[0133] 4. Lateral flow technology test results

[0134] The method for determining the test results of lateral flow technology is as follows: there is a bright band in the reading area of ​​the test strip test line of the positive control, no bright band in the reading area of ​​the test strip test line of the negative control, and a bright band in the reading area of ​​the quality control line. If there is a bright band in the reading area of ​​the test strip test line at 5 minutes, the test sample can be determined to be mycoplasma positive, otherwise it is negative.

[0135] like Fig.19 As shown, Fig.191-11 are the test strip banding results of oral mycoplasma (M.orale), hyorhinis, synoviae, fermentans, arginini, pneumoniae, A.laidlawii, S.citri, M.gallisepticum and M.salivarium at a concentration of 5 CFU / ml and the negative control sample at 5 min.

[0136] from Fig.19 It can be seen that when the lateral flow method is used to detect mycoplasma contamination in the test sample, compared with the negative control sample (the detection line of No. 11 does not show red), even if 10 types of mycoplasma at a concentration of 5 CFU / mL are mixed with MSC cells, very good detection results can be obtained at 3 minutes (the detection line shows red). This shows that the crRNA provided by the present invention is used for the lateral flow method to detect mycoplasma contamination, and 10 6 It has a very good detection effect on the mycoplasma concentration of 5 CFU / ml in each cell, and also meets the requirements of the alternative method for mycoplasma detection in the pharmacopoeia.

[0137] The present invention is also based on the better design ideas provided by Primer Explorer Version software, and adopts conventional primer design methods to design a detection primer and crRNA combination based on LAMP-CRISPR / Cas12a. Fig. 20 and 21 is the detection result of the combination of the detection primer and crRNA. Figure 20-21 It can be seen that compared with the present application, the primer and crRNA combination designed by the conventional design method targets a longer amplified fragment, has a low amplification efficiency, and cannot meet the sensitivity required by the pharmacopoeia (only 1000 copies / reaction). The types of mycoplasma required to be detected in the pharmacopoeia are relatively wide and the sequences are very different. If the conventional LAMP primer design scheme is applied to mycoplasma detection, it will be significantly limited.

[0138] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A combination of primers and crRNA for broad-spectrum detection of mycoplasma based on LAMP-CRISPR / Cas12a, characterized in that: It includes 2 outer primers, 5 inner primers, 4 loop primers and crRNA, the nucleotide sequences of the outer primers are shown in SEQ ID NOs.1 and 2, the nucleotide sequences of the inner primers are shown in SEQ ID NOs.3-7, and the nucleotide sequences of the loop primers are shown in SEQ ID NOs.8-11; the nucleotide sequences of the crRNA are shown in SEQ ID NOs.12-15, respectively; the primer pairs for preparing the crRNA are F1 / R1, F2 / R2, F3 / R3 and F4 / R4, and the nucleotide sequences of the primer pairs F1 / R1, F2 / R2, F3 / R3 and F4 / R4 are shown in SEQ ID NOs.16-23, respectively.

2. A LAMP-CRISPR / Cas12a system for detecting mycoplasma, characterized in that: The LAMP-CRISPR / Cas12a system includes the nucleotide sequences of the outer primers shown in SEQ ID NOs. 1 and 2, the inner primers shown in SEQ ID NOs. 3-7, and the loop primers shown in SEQ ID NOs. 8-11; The LAMP-CRISPR / Cas12a system also includes the crRNA shown in SEQ ID NO.12-15; or, it also includes primer pairs F1 / R1, F2 / R2, F3 / R3 and F4 / R4, and the nucleotide sequences of the primer pairs F1 / R1, F2 / R2, F3 / R3 and F4 / R4 are shown in SEQ ID NO.16-23, respectively.

3. The LAMP-CRISPR / Cas12a system according to claim 2, characterized in that The LAMP-CRISPR / Cas12a system also includes Cas12a protein, single-stranded DNA signal molecule, 10× buffer, RNasin and nuclease-free water.

4. A product for detecting mycoplasma, characterized in that: The product comprises the LAMP-CRISPR / Cas12a system of claim 2 or 3.

5. The product according to claim 4, characterized in that The product is a detection kit, a detection test strip or a detection chip.

6. A method for detecting mycoplasma not for the purpose of disease diagnosis and treatment, characterized in that: The test sample is pretreated, the genome of the test sample is extracted, the genome is used as a template for amplification, and the amplified product is used as a template, and the LAMP-CRISPR / Cas12a system described in claim 2 or 3, or the product described in claim 4 or 5 is used for detection and result determination.

7. The method according to claim 6, characterized in that The detection method is fluorescence or lateral flow.

8. The method according to claim 7, characterized in that The method for determining the result is as follows: when the fluorescence method is used, if the test sample has an obvious fluorescence growth curve and the fluorescence value is greater than 15 cfu, the test sample can be determined to be mycoplasma positive, otherwise it is negative; when the lateral flow method is used, if there is a bright band in the detection line reading area of ​​the test strip at 5 minutes, the test sample can be determined to be mycoplasma positive, otherwise it is negative.

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