CN115960902A

CN115960902A - CRISPR-Cas system-based primer for detecting various mycoplasmas, crRNA and application of CRISPR-Cas system-based primer

Info

Publication number: CN115960902A
Application number: CN202211607274.8A
Authority: CN
Inventors: 臧照星; 徐国东; 郝瑶; 罗航; 幸晓莹; 刘愈杰
Original assignee: Canvest Wuhan Biotechnology Co ltd
Current assignee: Canvest Wuhan Biotechnology Co ltd
Priority date: 2022-12-14
Filing date: 2022-12-14
Publication date: 2023-04-14
Anticipated expiration: 2042-12-14
Also published as: CN115960902B

Abstract

The invention discloses a primer for detecting various mycoplasmas based on a CRISPR-Cas system, crRNA and application thereof, and relates to the technical field of mycoplasma nucleic acid detection. The invention provides 4 pairs of primers capable of being used for preparing crRNA for detecting various mycoplasmas, and a CRISPR-Cas12a system constructed by mixing the crRNA prepared by the 4 pairs of primers can be used for detecting mycoplasma with 1 copy genome/reaction system or 2CFU/mL concentration. According to the result of durability analysisIt shows that the present invention can detect 10 ⁶ Mycoplasma with concentration of 2CFU/mL in each MSC cell, therefore, the culture method and the DNA staining method used by pharmacopoeia can be effectively replaced to detect mycoplasma; the method has the advantages of simple operation, short time, strong specificity, high sensitivity, wide coverage and the like, and is a relatively high-efficiency mycoplasma detection method at present.

Description

CRISPR-Cas system-based primer and crRNA for detecting various mycoplasmas and application thereof

Technical Field

The invention relates to the technical field of mycoplasma nucleic acid detection, in particular to a primer pair, crRNA and application thereof for detecting various mycoplasmas based on a CRISPR-Cas system.

Background

Mycoplasma is a group of the smallest prokaryotic cell type microorganisms that lack a cell wall, are highly polymorphic in morphology, and are able to propagate through a sterilizing filter and grow in an inanimate medium. Mycoplasma can cause various animal and plant diseases; for example, mycoplasma pneumoniae is a major cause of respiratory infections and pneumonia, ureaplasma urealyticum and mycoplasma hominis can cause urogenital infections, and spiroplasma can infect insects. Mycoplasma contamination is also a great threat to cell culture, and is likely to cause cell metabolism obstruction, surface structure damage, fusion force reduction and the like, thereby affecting therapeutic proteins of a host. According to the investigation data of the German DSMZ cell bank researchers, about 1/4 of the cells are affected by mycoplasma contamination. In the biopharmaceutical industry, the effects of mycoplasma contamination are almost devastating.

In view of the safety, purity and production efficiency of biological products, mycoplasma contamination detection is an important part of the quality control process of biological products. The corresponding supervision mechanism is also established by each national supervision department, and the general rule 3301 of the Chinese pharmacopoeia makes clear regulations on each production link of biological products. The European Union pharmacopoeia 2.6.2 indicates that the main cell bank, the working cell bank, the virus seed batch and the reference cell use a culture method and a cell indication method to detect mycoplasma; some viral harvests and vaccine products are cultured to detect mycoplasma contamination. The United states pharmacopoeia Commission 63 states that mycoplasma contamination detection is an essential quality control requirement to ensure the reliability of biological products and materials used in their production, and the Japanese pharmacopoeia part G3 states that mycoplasma detection is performed on cell matrices for biological products.

At present, the main methods for monitoring mycoplasma pollution are a culture method and an indicator cell method, and the two methods have long monitoring period and large workload. With the increasing variety of biological products, especially the advent of leading-edge therapeutic drugs based on gene therapy, cell therapy or tissue engineering, the shortcomings of cell culture methods and indicator cell detection methods are more evident. Compared with biomacromolecule biological products, most therapeutic products cannot be sterilized, have short validity period and low batch yield, and need shorter detection period. Therefore, a fast-releasing mycoplasma contamination detection method is added to pharmacopoeias in many countries. For example, the european union pharmacopoeia and japanese pharmacopoeia indicate that Nucleic acid amplification technologies (NAT) or other appropriately validated technologies can be used in place of the culture method and the indicator cell method, and that sensitivity and specificity of detection by the NAT technology are clearly specified: when the mycoplasma is detected by a quasi-substitution culture method, the sensitivity of NAT detection is 10CFU/mL; when the DNA staining method is to be replaced, the sensitivity of NAT detection should reach 100CFU/mL. The united states pharmacopoeia specifies that NAT technology can be used to detect mycoplasma instead of culture and indicator cell methods. The Chinese pharmacopoeia also indicates that in addition to the culture method and the indicator cell method for detecting mycoplasma, other methods approved by the national drug certification institute can be used.

The CRISPR-Cas (Clustered regular interleaved Short Palindromic Repeats, CRISPRs) system is a self-defense system evolved in the fight between bacteria and viruses. At present, the CRISPR-Cas system is widely used In Vitro Diagnosis (IVD), and more common protein families comprise CRISPR-Cas12, CRISPR-Cas13, CRISPR-Cas14 and the like. The CRISPR-Cas12a protein is a DNA-targeting nuclease guided by crRNA that is capable of binding to and cleaving the corresponding DNA strand. When the CRISPR-Cas12a protein is combined with a target DNA which is complementary to crRNA and contains a PAM region, the cis-form DNase activity of the CRISPR-Cas12a protein is activated, the target DNA is cut, and simultaneously, the adjacent linear or circular single-stranded DNA molecule can be completely degraded.

Disclosure of Invention

The invention designs a set of method for efficiently targeted detection of multiple mycoplasma 23s rRNA genes in biological products based on the functional characteristics of CRISPR-Cas12a protein, which comprises primers for preparing crRNA and corresponding crRNA, and a method and a detection kit for detecting multiple mycoplasma pollution in biological products by using the crRNA. Based on the specificity and signal amplification of the two technologies of PCR amplification and CRISPR-Cas12a protein detection, the invention is equivalent to twice confirmation and twice signal amplification of a target to be detected, and the specificity, sensitivity and coverage of detection are obviously improved. The technology provided by the invention can be combined with the DNA Barcoding technology to realize the identification of the pollution mycoplasma; the visual detection of mycoplasma can be realized by using a lateral flow detection technology; the method is simple to operate, reduces the requirements of instruments and the cost, and is a high-efficiency mycoplasma identification and detection method. Specifically, this is achieved by the following technique.

A primer for preparing crRNA for detecting various mycoplasmas comprises a primer pair F1, a primer pair F2, a primer pair F3 and a primer pair F4;

the forward primer sequence (shown as SEQ ID NO. 1) of the primer pair F1 is as follows:

5’-taatacgactcactatagggtaatttctactaagtgtagatattatagtcagtacggctagg-3’；

the reverse primer sequence (shown as SEQ ID NO. 2) of the primer pair F1 is as follows:

5’-cctagccgtactgactataatatctacacttagtagaaattaccctatagtgagtcgtatta-3’；

the forward primer sequence (shown as SEQ ID NO. 3) of the primer pair F2 is as follows:

5’-taatacgactcactatagggtaatttctactaagtgtagatattgtattcagtacgcctagg-3’；

the reverse primer sequence (shown as SEQ ID NO. 4) of the primer pair F2 is as follows:

5’-cctaggcgtactgaatacaatatctacacttagtagaaattaccctatagtgagtcgtatta-3’；

the forward primer sequence (shown as SEQ ID NO. 5) of the primer pair F3 is as follows:

5’-taatacgactcactatagggtaatttctactaagtgtagatattgagatcagtaccccgagg-3’；

the reverse primer sequence (shown as SEQ ID NO. 6) of the primer pair F3 is as follows:

5’-cctcggggtactgatctcaatatctacacttagtagaaattaccctatagtgagtcgtatta-3’；

the forward primer sequence (shown as SEQ ID NO. 7) of the primer pair F4 is as follows:

5’-taatacgactcactatagggtaatttctactaagtgtagatattgcattcagtacccctagg-3’；

the reverse primer sequence (shown as SEQ ID NO. 8) of the primer pair F4 is as follows:

5’-cctaggggtactgaatgcaatatctacacttagtagaaattaccctatagtgagtcgtatta-3’。

the invention provides a preparation method of crRNA for detecting various mycoplasmas, which comprises the following steps:

s1, synthesizing an in vitro transcription template of the crRNA by using the primers, namely the primer pairs F1, F2, F3 and F4;

and S2, taking the in vitro transcription template prepared in the step S1 as a template, carrying out in vitro transcription, and then recovering the crRNA.

In general, the methods for synthesizing in vitro transcription templates and in vitro transcription of steps S1, S2 described above can be performed using methods, reagents and reaction condition parameters conventional in the art. For example, step S1 may be performed by passing the reaction product through a collection column using a commercially available DNA recovery kit, and collecting the template by repeating centrifugation and washing several times according to the instructions of the kit; step S2 can use a commercial in vitro transcription kit to perform reaction according to the instruction of the kit, then the in vitro transcription is completed by passing through a collection column and centrifugal separation and purification, finally the conventional operation is performed by using a commercial RNA recovery kit according to the instruction of the kit, and the crRNA is separated and recovered by the steps of passing through the collection column, centrifugal separation and purification, elution and the like.

Preferably, in step S1, the transcription reaction system is: mu.L of nucleic acid-free water, 5. Mu.L of each forward primer of 10. Mu.M of the primer pairs F1, F2, F3 and F4, 5. Mu.L of each reverse primer of 10. Mu.M of the primer pairs F1, F2, F3 and F4, and 10. Mu.L of 5 Xannealing buffer; the transcription reaction program was started from 95 ℃ to 25 ℃ at a rate of 0.1 ℃/s. The reaction system and the reaction program can be carried out in an amplification instrument, and the high-efficiency transcription reaction can be ensured along with the slow temperature reduction in the amplification instrument, so that as many in vitro transcription templates as possible can be obtained.

The invention also provides a CRISPR-Cas12a system for detecting various mycoplasmas, which comprises the crRNA prepared by adopting the method.

Preferably, the system further comprises a Cas12a protein, a single-stranded DNA signal molecule, a 10 × buffer, RNasin, and a nucleic acid-free water. Cas12a protein, single-stranded DNA signal molecule, 10 × buffer solution, RNase and nucleic acid-free water are all commonly used reagent raw materials in CRISPR-Cas12a technology, and can be purchased from commercial sources. As long as CRISPR-Cas12a treatment and detection are carried out by using the crRNA provided by the invention, a very good detection effect can be obtained for various mycoplasmas in biological products by using any Cas12a protein, single-stranded DNA signal molecules, 10 Xbuffer solution, RNase and nucleic acid-free water which are sold on the market.

The invention also provides a method for detecting various mycoplasma, which comprises the following steps:

p1, pretreating a sample to be detected, and extracting a genome of the sample to be detected;

p2, taking the genome of the sample to be detected prepared in the step P1 as a template for amplification, and recovering an amplification product;

and P3, mixing the components of the CRISPR-Cas12a system by taking the amplification product recovered in the step P2 as a template, detecting by adopting a fluorescence method or a lateral flow method, and judging the result.

In general, steps P1-P3 are similarly performed using conventional methods and kits in the art. For example, in the step P1, the sample to be tested is pretreated by conventional centrifugation, separation, and the like, so as to control the density of cells in the working solution of the sample to be tested, and the cell can be directly used after preparation, or can be stored at low temperature for later use; the genome can be extracted by using a commercially available kit according to the kit instructions. The amplification in step P2 can be processed by using a conventional kit, or primers can be designed, reagents can be prepared, or assay parameters can be adjusted, and primers, reagents, and assay parameters with the best amplification effect can be selected by a large number of assays. In step P3, the detection method may be fluorescence detection method or lateral flow detection method, or other detection methods commonly used in the art may be used.

Preferably, in step P2 of the detection method, the sequence of the amplification forward primer used for amplification is as follows:

5 '-glass-fiber-containing ggtagagagcactgaatatggaatggaatggc-fiber-containing 3' (shown as SEQ ID NO. 9);

5 'and ggtaaaagctactgaatgtatgatggc-3' (shown as SEQ ID NO. 10);

5 '-glass-doped ggtagagagcctgaatgtatgatggc-doped 3' (shown as SEQ ID NO. 11);

the amplification reverse primers used were identical to the amplification forward primers described above, and had the following sequences:

5 '-ctcgacwaggctttacgcac-3' (shown in SEQ ID NO. 12).

The invention specially designs the primers for the amplification of the genome of a sample to be detected, and selects and obtains the three groups of primers from a large number of primers through a large number of comparison experiments. The three groups of primers are simultaneously adopted in an amplification system, so that the high-efficiency detection of more mycoplasma-like organisms can be realized.

Preferably, in step P3 of the detection method, the result determination method specifically includes: when the fluorescence method is adopted for detection, if the detected sample has a fluorescence increase curve and the fluorescence value is more than 15cfu (the threshold value is determined based on the curve method in Chinese pharmacopoeia), the detected sample is judged to be mycoplasma positive, otherwise, the detected sample is negative. When the threshold is below 15cfu, the detection result is prone to false positives.

When the lateral flow method is adopted for detection, if the test strip detection line area after reacting for 3min has a bright band, the sample to be detected is judged to be mycoplasma positive, otherwise, the sample to be detected is negative.

The invention also provides a kit for detecting various mycoplasmas, which contains the primers for preparing the crRNA for detecting various mycoplasmas, or contains the crRNA, or contains the CRISPR-Cas12a system.

Compared with the prior art, the invention has the advantages that:

1. according to the primer pair, the crRNA and the corresponding method for detecting multiple mycoplasma in a sample, provided by the invention, a novel CRISPR-Cas12a protein crRNA targeting site is designed in a mycoplasma 23s rRNA gene amplification fragment, a CRISPR-Cas12a protein detection system is used for carrying out signal amplification once after amplification signals of a sample to be detected are amplified, and compared with a common PCR detection technology, a culture method and a DNA dyeing method in pharmacopoeia, the method has the advantages of short time, simplicity in operation, low price, strong specificity, high sensitivity and the like;

2. compared with a fluorescent quantitative PCR detection method, the method provided by the invention has the advantages that although the PCR process is also available, the method is only carried out for 35 cycles, the cycle process has no fluorescent reading stage, the time is about 70min when the subsequent fluorescent detection stage is used together, the time is shorter, and the sensitivity is far higher than that of the fluorescent quantitative detection.

3. In the process of detecting mycoplasma, when the PCR technology is used for amplifying the mycoplasma fragment, compared with RPA amplification, the requirements on primers are lower, the fault tolerance of base mismatching is strong, the amplification efficiency is high, and the high-sensitivity and high-coverage mycoplasma detection can be realized;

4. the method can be combined with a DNA barcode technology, and compared with LAMP amplification, the method can realize the identification of the polluted mycoplasma; only one pair of amplification primers is needed, the coverage rate of the primers is higher, and the detection range is wider.

Drawings

FIG. 1 shows the results within 40min of the fluorescence test of the detection specificity of the present invention using bacteria. In the figure, no.1 is a positive control sample, and nos. 2 to 12 are clostridium acetobutylicum (c.acetobutylicum), streptococcus pneumoniae (s.pneumoniae), lactobacillus acidophilus (l.acidophilus), escherichia coli (e.coli), staphylococcus aureus (s.aureus), clostridium sporogenes (c.sponogenes), bacillus subtilis (b.subtilis), mycobacterium phlei (m.ph), pseudomonas aeruginosa (p.aeruginosa), micrococcus luteus (m.luteus) sample and negative control (NTC), respectively;

FIG. 2 shows the results of the fluorescence test of the detection specificity of the present invention using bacteria at 40 min. In the figures, the numbers 1 to 12 have the same meaning as in FIG. 1;

FIG. 3 shows the results within 40min of the fluorescence test of the detection specificity of the present invention using fungi. 1 is a positive control sample, 2-4 are aspergillus niger (a. Niger), candida albicans (c. Albicans) and negative control (NTC), respectively;

FIG. 4 shows the results of the detection specificity of the present invention at 40min, which was verified by the fluorescence method using fungi; in the figures, the numbers 1 to 4 have the same meaning as in FIG. 3;

FIG. 5 shows the results within 40min of the fluorescence verification of the detection specificity of the present invention using cells commonly used for the production of biologicals; in the figure, the number 1 is a positive control, and the numbers 2 to 12 are cell line samples and negative controls (NTC) which are commonly used in the production of Hi-5, sf9, A9, 293, CHO-K1, MSC, vero, MDBK, PK-15 and Walker-256 biological products respectively;

FIG. 6 shows the results of the fluorescence test of the specificity of the assay of the present invention using cells commonly used in the production of biologicals at 40 min; in the figures, the numbers 1 to 12 have the same meaning as in FIG. 5;

FIGS. 7-16 show the results of the sensitivity verification of the present invention. FIGS. 7-16 are

assays

1, 5, 10, respectively ² 、10 ³ 、10 ⁴ And 10 ⁵ Fluorescence changes of 10 mycoplasma, m.orale, m.hyornis, m.synoviae, m.salivariam, m.prefermentans, m.arginini, m.pneumniae, a.laidlawii, s.citri, m.gallisepticum, of copy/reaction;

figure 17 is the pharmacopoeia alternative verification result of the present invention; in the figure, numbers 1-10 are respectively 2CFU/mL concentration of M.orale, M.hyornis, M.synoviae, M.fermentans, M.arginini, M.pneumoniae, A.laidlawii, S.citri, M.gallisepticum, 9 kinds of mycoplasmas, and negative control within 40min of fluorescence detection results;

FIG. 18 is the pharmacopoeia substitutability verification results of the present invention; in the figure, numbers 1-10 are respectively 2CFU/mL concentration of M.orale, M.hyornis, M.synoviae, M.fermentans, M.arginini, M.pneumoconiae, A.laidlawii, S.citri, M.gallisepticum 9 mycoplasma, and negative control at 5min fluorescence detection;

FIG. 19 is a durability analysis of the present invention. In the figure, numbers 1 to 10 are M.orale, M.hyornis, M.synoviae, M.formenans, M.arginini, M.pneumoniae, A.laidlawii, S.citri, M.gallisepticum and 10CFU/ml concentrations, respectively ⁶ The fluorescence dynamics detection results of the MSC cell mixed sample with copy number and the single MSC cell sample within 40 min;

FIG. 20 is a durability analysis of the present invention. In the figure, numbers 1-10 are M.orale, M.hyornis, M.synoviae, M.prefermentans, M.arginini, M.pneumoconiae, A.laidlawii, S.citri, M.gallisepticum and 10CFU/ml concentrations, respectively ⁶ Fluorescence detection results of the MSC cell mixed sample and the single MSC cell sample at 5 min;

FIG. 21 shows the results of the lateral flow technique for detecting Mycoplasma according to the present invention. In the figure, numbers 1 to 10 are M.orale, M.hyornis, M.synoviae, M.formenans, M.arginini, M.pneumoniae, A.laidlawii, S.citri, M.gallisepticum and 10CFU/ml concentrations, respectively ⁶ Results of 3min detection of the mixed MSC cell sample and the individual MSC cell sample.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following detailed description, reagents and equipment used are commercially available in the ordinary market unless otherwise specified. To verify the effect of the present invention, various mycoplasma MB standards (mycoplasma detected as indicated in the european pharmacopoeia) were used as positive controls.

Example 1: pretreatment of the sample to be tested, positive control and negative control, extraction of genomic DNA of the sample to be tested

1. Pretreatment of samples to be tested (purchased from MB Corp.)

And centrifuging the sample to be detected, collecting the precipitate for use, or directly using the precipitate, or storing the precipitate at-80 ℃ for later use.

2. Pretreatment of positive and negative controls

Positive control: a mixture of 200 copies of the genome of Mycoplasma oralis and 200 copies of the genome of Mycoplasma pneumoniae was added to the MB Mycoplasma standard, and the mixture was treated in the same manner as the test sample.

Negative control: 1mL of MEM basal medium containing 10% FBS was treated in the same manner as the sample to be tested.

3. Extraction of genome of sample to be tested (relevant reagent and kit purchased from health to Bio corporation)

The DNA extraction kit provided by Kangyi biological company is used for extracting the genome of a sample to be detected according to the requirements of the kit specification, and the genome is dissolved in 100 mu L of RNA-free water.

Example 2: preparation of cr-RNA

1. Design of primers for preparation of cr-RNA

Utilizing MEGA5.0 software, comparing 16s rRNA-23s rRNA genes of 21 common mycoplasma and other gram-positive bacteria (indicated by pharmacopoeia), designing a plurality of pairs of primers in a region with mycoplasma gene sequence specificity according to conservation among the mycoplasma and difference between the mycoplasma and other gram-positive bacteria, obtaining four crRNAs with optimal specificity and sensitivity through screening, and designing 4 primer pairs F1, F2, F3 and F4 for synthesizing the crRNAs, wherein forward primer sequences and reverse primer sequences of the primer pairs F1, F2, F3 and F4 are respectively as follows:

5’-taatacgactcactatagggtaatttctactaagtgtagatattatagtcagtacggctagg-3’；

5’-cctagccgtactgactataatatctacacttagtagaaattaccctatagtgagtcgtatta-3’；

5’-taatacgactcactatagggtaatttctactaagtgtagatattgtattcagtacgcctagg-3’；

5’-cctaggcgtactgaatacaatatctacacttagtagaaattaccctatagtgagtcgtatta-3’；

5’-taatacgactcactatagggtaatttctactaagtgtagatattgagatcagtaccccgagg-3’；

5’-cctcggggtactgatctcaatatctacacttagtagaaattaccctatagtgagtcgtatta-3’；

5’-taatacgactcactatagggtaatttctactaagtgtagatattgcattcagtacccctagg-3’；

5’-cctaggggtactgaatgcaatatctacacttagtagaaattaccctatagtgagtcgtatta-3’。

2. synthesis of in vitro transcription templates

The primer pairs F1, F2, F3 and F4 were selected to prepare the reaction system shown in Table 1 below.

TABLE 1 reaction System for the Synthesis of in vitro transcription templates

Nuclease-free water	30μL
		Forward primer of primer pair F1/F2/F3/F4 (10. Mu.M)	5μL
Reverse primer of primer pair F1/F2/F3/F4 (10. Mu.M)	5μL
		5 × annealing buffer	10μL
Total	50μL

The transcription reaction program was started from 95 ℃ to 25 ℃ in a PCR instrument at a rate of 0.1 ℃/s.

3. Recovery of in vitro transcription templates Using DNA recovery kit (NEB, D4033)

(1) Adding DNA binding buffer with 5 times volume into the reaction product, and uniformly mixing the mixed solution;

(2) Adding the above solution to Zymo-Spin ^TM In a Col mu Mn2 collecting column, 8000g of the mixture is centrifuged for 30s; adding 200 mu.L of DNA wash buffer, centrifuging for 30s at 9000g, and repeating once again; discarding the filtrate; putting the collection column into the centrifuge again, and centrifuging at 12000g for 3min;

(3) mu.L of RNA-free water was added to the collection column, incubated at room temperature for 2min, and centrifuged at 9000g for 2min to collect the template.

4. In vitro transcription Using in vitro transcription kit (NEB, E2050S)

The collected in vitro transcription templates were selected as reaction templates to prepare the reaction system shown in Table 2 below.

TABLE 2 in vitro transcription reaction System

Nuclease-free water	10μL
		NTP Buffer Mix	10μL
Template DNA (Template DNA)	8μL
		T7 RNA Polymerase Mix	2μL
Total	30μL

After mixing, the mixture was centrifuged briefly, incubated at 37 ℃ for 5 hours, then 45. Mu.L of nucleic-free water was added, followed by 2. Mu.L of DNase I (DNA hydrolase) and mixed well, and incubated at 37 ℃ for 20 minutes to obtain a transcript.

5. Recovery of cr-RNA Using an RNA recovery kit (NEB, T2040L)

(1) Adding 2 times of RNA clean Binding Buffer into the transcription product, and uniformly mixing the mixed solution; adding equal volume of anhydrous ethanol into the solution, mixing gently without vortex, and standing for 1-2min;

(2) Adding the above solution into a collecting column, and centrifuging at 8000g for 1min; the filtrate was discarded, 500. Mu.L of RNA clean Wash Buffer 9000g was added and centrifuged for 1min (repeated once); discarding the filtrate, putting the column into the centrifuge again, and centrifuging at 12000g for 3min;

(3) Adding 50 μ L RNA-free water into the collection column, centrifuging at 9000g for 2min to collect crRNA, and preparing the final crRNA sequence shown in SEQ ID NO.13-16 by using four primer pairs F1, F2, F3, and F4.

The crRNA sequence finally prepared by the primer pair F1 (shown as SEQ ID NO. 13) is as follows:

uaauuucuacuaaguguagauauuauagucaguacggcuagg

the reverse primer sequence (shown as SEQ ID NO. 14) of the primer pair F1 is as follows:

uaauuucuacuaaguguagauauuguauucaguacgccuagg

the forward primer sequence (shown as SEQ ID NO. 15) of the primer pair F2 is as follows:

uaauuucuacuaaguguagauauugagaucaguaccccgagg

the reverse primer sequence (shown as SEQ ID NO. 16) of the primer pair F2 is as follows:

uaauuucuacuaaguguagauauugcauucaguaccccuagg；

in the above-mentioned SEQ ID NO.13-16, U represents uracil, and U is replaced with T in the submitted electronic sequence Listing as required by the WIPO standard.

Example 3: PCR amplification of the genome of a sample to be tested

1. Primer design for PCR amplification

Utilizing MEGA5.0 software to compare 16s rRNA-23s rRNA genes of common 21 mycoplasma and other gram-positive bacteria (indicated by pharmacopeia), designing a plurality of primers in a region of mycoplasma gene sequence specificity according to conservation among the mycoplasma and difference between the mycoplasma and other gram-positive bacteria, and obtaining a group of amplification primer pairs with optimal specificity and sensitivity by screening, wherein the forward primer sequences are respectively as follows:

5 '-glass-fiber-doped ggtagagagcctgaatatgaatggc-doped 3' (shown as SEQ ID NO. 10);

5 'ggtaaaagctactgaatgtatgatggc-3' (shown as SEQ ID NO. 11);

5 '-glass-doped ggtagagagcctgaatgtatgatggc-doped 3' (shown as SEQ ID NO. 12);

the amplification reverse primer sequence of the amplification primer pair is as follows:

5 '-ctcgacwaggctttacgcac-3' (shown in SEQ ID NO. 13)

2. Amplification reaction process

The PCR amplification reaction system shown in Table 3 below was configured using the Champagne Taq DNA Polymerase (Novowed) kit and the above three sets of amplification primer pairs.

TABLE 3 reaction System for the Synthesis of in vitro transcription templates

Nuclease-free water	9.1μL
		10 XPCR buffer	2.5μL
Template DNA	10μL
		Forward primer (10. Mu.M)	Each of the three forward primers is 0.4. Mu.L
Reverse primer (10. Mu.M)	1.2μL
		DNA polymerase	0.5μL
Total	25μL

The PCR amplification reaction procedure is shown in Table 4 below.

TABLE 4 PCR amplification reaction procedure

And collecting PCR amplification reaction products.

Example 4: the present example used various bacteria such as three gram-positive bacteria (clostridium acetobutylicum (c), streptococcus pneumoniae(s), lactobacillus acidophilus (l), escherichia coli (e), staphylococcus aureus(s), clostridium sporogenes (c), bacillus subtilis (b), mycobacterium phlei (m.ph), pseudomonas aeruginosa (p.aeruginosa), micrococcus luteus (m.luteus)), fungi (aspergillus niger (a.niger) and candida albicans (c.albicans)) and cell lines of biological products (Hi-5, sf9, A9, 293, CHO-K1, MSC, vero, MDBK, PK-15, walker-256 cell lines) as indicated by the pharmacopoeia, and the specificity detection of the present invention was performed by the fluorescence method and the lateral flow method, respectively.

1. Detection by fluorescence

The fluorescence detection reaction system is shown in the following table 5:

TABLE 5 detection of reaction systems by fluorescence method

Nuclease-free water	32.5μL
		NEBbuffe2.1r(NEB，B6002V)	6μL
RNase inhibitor(TaKaRa，2311A)	0.5μL
		crRNA(2μM)	2μL
2FAM-ssDNA-Biotin(5μM)	2μL
		Form panel	15μL
Cas12a protein (Tu Luo gang biology, 32108-01)	2μL
		Total	60μL

Fluorescence detection was performed using a multifunctional microplate reader (srefei, varioskan LUX). The mode is kinetic detection, the temperature is 38 ℃, the excitation wavelength is 485nm, and the emission wavelength is 520nm.

FIGS. 1 and 2 show the results of the fluorometric test of the detection specificity of the present invention using bacteria within 40min and at 40min, respectively. As can be seen from FIGS. 1 and 2, the fluorescence intensity curve of the positive control of number 1 rapidly increased, and reached a maximum of 400 at about 20min, and then remained substantially unchanged by 40 min; the fluorescence intensity curves of numbers 2-12 are basically overlapped within 0-40min and are all basically horizontal straight lines, and the fluorescence intensity is always close to 0. This indicates that the detection result of the mycoplasma positive control using the crRNA of the present invention has significant specificity relative to common bacteria.

FIGS. 3 and 4 show the results of the fluorometric detection of the specificity of the detection of the present invention using 2 common fungi at 40min and 40min, respectively. As can be seen from FIGS. 3 and 4, the fluorescence intensity curve of the positive control No.1 rapidly increased, and reached a maximum of 400 at about 20min, and then remained substantially unchanged by 40 min; the fluorescence intensity curves of numbers 2-4 are completely overlapped within 0-40min and are all basically horizontal straight lines, and the fluorescence intensity approaches to 0 all the time. This indicates that the detection result of the mycoplasma positive control using the crRNA of the present invention has significant specificity relative to the common 2 fungi.

FIGS. 5 and 6 show the results of fluorescence tests of the specificity of the assay of the present invention using 10 commonly used cell lines at 40min and 40min, respectively; as can be seen from FIGS. 5 and 6, the fluorescence intensity curve of the positive control No.1 rapidly increased, and reached a maximum of 400 at about 20min, and then remained substantially unchanged by 40 min; the fluorescence intensity curves of numbers 2-12 are completely overlapped within 0-40min and are all basically horizontal straight lines, and the fluorescence intensity approaches to 0 all the time. This demonstrates that the detection results of the mycoplasma positive control using the crRNA of the present invention have significant specificity relative to the common 10 cell lines.

As shown in FIGS. 7 to 16, it can be seen that the fluorescence intensity of 10 Mycoplasma 1 copy/reaction rapidly increased in the range of 0 to 40min, and the final fluorescence intensity reached 400 or more. This indicates that the crRNA provided by the present invention also has very good sensitivity to 1 copy of Mycoplasma.

As can be seen from fig. 17 and 18, compared with the negative control, the fluorescence intensity of 9 common mycoplasma with a concentration of 2CFU/mL is significantly increased within 40min and reaches a maximum value of more than 400 by using the crRNA provided by the present invention, and the fluorescence intensity at 5min is also significantly higher than that of the negative control, which indicates that the method of the present invention shows very significant specificity for mycoplasma detection, and can effectively replace the standard method in pharmacopoeia.

It can be seen from figures 19, 20 that even 9 common mycoplasma at a concentration of 2CFU/mL were compared to 10 MSC cell samples alone ⁶ The MSC cells were mixed and were also found to have a significant increase in fluorescence intensity within 40min, with very high fluorescence intensity already reached at 5 min. This demonstrates that the method of the present invention is highly robust and suitable for the detection of samples in which high concentrations of cells are mixed with mycoplasma. While having a very high sensitivity.

2. Lateral flow assay

The lateral flow assay reaction system is shown in table 6 below:

TABLE 6 lateral flow assay reaction systems

And (3) putting the reaction system into a thermostat with the temperature of 38 ℃ for reaction, putting the nucleic acid detection test strip special for Cas12 (M20801-F007) into the reaction system after 10min, observing the result after 3min, and taking a picture.

As can be seen from FIG. 21, when the lateral flow method was used to detect Mycoplasma contamination in MSC cell preparations, even 9 Mycoplasma at a concentration of 2CFU/mL and 10 Mycoplasma were compared to MSC cell samples alone (no red coloration on the detection line) ⁶ The MSC cells are mixed, and a very good detection result can be obtained at 3min (the detection line shows red). This shows that the use of the crRNA provided by the invention in lateral flow method for detecting mycoplasma contamination can detect 10 ⁶ The mycoplasma with the concentration of 2CFU/ml in each MSC cell has very good detection effect on a sample mixed with the cells and the mycoplasma, and meets the requirement of a mycoplasma detection substitution method in pharmacopoeia.

In conclusion, based on the above detection results, and the comparison and search with the NCBI database and the Silva database with high similarity, it can be scientifically and reasonably predicted that the present invention can also effectively detect more than 140 mycoplasma, including mycoplasma types specified in pharmacopoeia of various countries and mycoplasma types of common contaminated cells.

The practice of the present invention has been described in detail with reference to the foregoing detailed description, but the invention is not limited to the specific details of the foregoing embodiment. Various simple modifications and changes can be made to the technical solution of the present invention within the scope of the claims and the technical idea of the present invention, and these simple modifications belong to the protection scope of the present invention.

Claims

1. A primer for preparing crRNA for detecting various mycoplasmas is characterized by comprising a primer pair F1, a primer pair F2, a primer pair F3 and a primer pair F4; the forward primer of the primer pair F1 is shown as SEQ ID NO.1, and the reverse primer is shown as SEQ ID NO. 2; the forward primer of the primer pair F2 is shown as SEQ ID NO.3, and the reverse primer is shown as SEQ ID NO. 4; the forward primer of the primer pair F3 is shown as SEQ ID NO.5, and the reverse primer is shown as SEQ ID NO. 6; the forward primer of the primer pair F4 is shown as SEQ ID NO.7, and the reverse primer is shown as SEQ ID NO. 8.

2. A preparation method of crRNA for detecting various mycoplasmas is characterized by comprising the following steps:

s1, synthesizing an in vitro transcription template of the crRNA by using the primer of claim 1, namely the primer pair F1, F2, F3 and F4;

and S2, taking the in vitro transcription template prepared in the step S1 as a template, carrying out in vitro transcription, and then recovering a product, namely the crRNA.

3. The method for preparing crRNA according to claim 2, wherein in step S1, the transcription reaction system is: mu.L of nucleic acid-free water, 5. Mu.L of each forward primer of 10. Mu.M of the primer pairs F1, F2, F3 and F4, 5. Mu.L of each reverse primer of 10. Mu.M of the primer pairs F1, F2, F3 and F4, and 10. Mu.L of 5 Xannealing buffer; the transcription reaction program was started from 95 ℃ to 25 ℃ at a rate of 0.1 ℃/s.

4. A CRISPR-Cas12a system for detecting multiple mycoplasmas comprising crRNA prepared by the preparation method of claim 2 or 3.

5. The CRISPR-Cas12a system according to claim 4, further comprising Cas12a protein, single-stranded DNA signal molecule, 10 x buffer, RNase and no nucleic acid.

6. A method for detecting various mycoplasmas is characterized by comprising the following steps:

p1, preprocessing a sample to be detected, and extracting a genome of the sample to be detected;

p2, amplifying by taking the genome of the sample to be detected prepared in the step P1 as a template, and recovering an amplification product;

and P3, mixing the components of the CRISPR-Cas12a system of claim 4 or 5 by taking the amplification product recovered in the step P2 as a template, and carrying out detection and result judgment.

7. The method for detecting various mycoplasmas according to claim 6, wherein in step P2, the amplification forward primers used for amplification have the sequences shown as SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11, and the amplification reverse primers have the sequences shown as SEQ ID No. 12.

8. The method for detecting multiple mycoplasmas according to claim 6, wherein in step P3, the method for determining the result is specifically: when the fluorescence method is adopted for detection, if the detected sample has a fluorescence increase curve and the fluorescence value is more than 15cfu, the sample to be detected is judged to be mycoplasma positive, otherwise, the sample to be detected is negative;

9. A kit for detecting multiple mycoplasma comprising the primer of claim 1, or comprising crRNA prepared by the preparation method of claim 2 or 3, or comprising the CRISPR-Cas12a system of claim 4 or 5.