Primer, probe, and kit for broad-spectrum detection of mycobacteria and use thereof

Abstract

Provided are a primer, probe, and kit for broad-spectrum detection of mycobacteria and a use thereof, relating to the technical field of broad-spectrum detection of mycobacteria. A primer-probe combination comprises a forward primer, a reverse primer and a probe. The nucleotide sequence of the forward primer is shown in SEQ ID NOs. 1 and 2, the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 3, and the nucleotide sequence of the detection probe is shown in SEQ ID NOs. 4-6. The primer-probe combination can detect more than 160 types of mycobacteria, covering a wide range; the detection time is greatly shortened; the present invention has strong specificity, and has no cross reaction with non-mycobacterial cells and viral genomes; the present invention has high detection sensitivity, with the detection limit reaching 100 CFU / ml and the genome detection limit being 10-20 copies / reaction; the present invention has high durability and high resistance to matrix interference.

Description

Primers, probes, kits for broad-spectrum detection of mycobacteria and their applications Technical Field

[0001] The present invention relates to the technical field of broad-spectrum detection of mycobacteria, and in particular to primers, probes, a kit and applications thereof for broad-spectrum detection of mycobacteria. Background Art

[0002] Mycobacteria are a group of slender, slightly curved rods without flagella and spores, growing in a branching pattern. Mycobacteria are divided into three major groups: Mycobacterium tuberculosis complex (MTC), Mycobacterium leprae, and nontuberculous mycobacteria (NTM). According to the LPSN (https: / / lpsn.dsmz.de / ), there are currently 266 species and 24 subspecies of mycobacteria. Mycobacterial infections cause chronic diseases accompanied by granulomas, such as tuberculosis and leprosy, which pose a significant threat to human health. Nontuberculous mycobacteria, which are widely present in environmental water and soil, are opportunistic pathogens and can cause lung infections, lesions in tissues and organs such as lymph nodes, bones, joints, skin, and soft tissues, and even systemic disseminated disease. In recent years, the prevalence of infectious diseases caused by nontuberculous mycobacteria has increased worldwide. An increasing number of nontuberculous mycobacteria have been reported to be pathogenic and potentially harmful. Currently, about 50 pathogenic mycobacterial species have been reported to be isolated from clinical and environmental sources.

[0003] The National Medical Products Administration and other regulatory agencies require that cell banks, cell therapy biological products, and other products be tested for mycobacterial contamination. Conventional methods for detecting mycobacteria are culture or guinea pig methods. The culture method involves inoculating the sample to be tested onto solid culture medium and incubating it at 37°C for 56 days. The guinea pig method requires the guinea pig to be autopsied 42 days after inoculation. Both the culture and guinea pig methods suffer from long testing cycles, slow growth, low isolation rates, and susceptibility to contamination. These methods cannot meet the needs for rapid testing and release of stem cell therapy biological products, which require high timeliness and sample costs. FDA guidelines, the European Pharmacopoeia, and the Chinese Pharmacopoeia clearly specify the use of fully validated nucleic acid amplification (NAT) methods for detecting mycobacteria. Therefore, developing a simple, efficient, reproducible, and sensitive broad-spectrum detection method based on the technical principles of the NAT method, specifically targeting the vast majority of mycobacteria, is an urgent need for the development of mycobacterial detection technology in my country.

[0004] Summary of the Invention

[0005] The qPCR method within the NAT method offers advantages such as simplicity, efficiency, good reproducibility, and high sensitivity. It is widely used for quality control testing of biological products both domestically and internationally. This invention has developed a TaqMan fluorescent quantitative PCR primer-probe combination, kit, and detection method for rapid and broad-spectrum detection of over 160 mycobacterial species. This method can detect mycobacterial contamination within a few hours, significantly meeting the demand for timely testing of biological products such as cell banks, virus seed banks, and cell therapy products. This is achieved specifically through the following technologies.

[0006] A primer-probe combination for broad-spectrum detection of mycobacteria comprises a forward primer, a reverse primer, and a probe. The nucleotide sequences of the forward primer are shown in SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3, and the nucleotide sequences of the detection probe are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6. The 5' end of the detection probe is coupled to a first fluorescent group, and the 3' end is coupled to a quenching group.

[0007] Preferably, the first fluorescent group is a FAM, TET, NED, ROX, CY3, CY5, VIC, JOE or HEX fluorescent group; and the quencher group is a TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1 or BHQ2 quencher group.

[0008] Preferably, the first fluorescent group is a FAM fluorescent group, and the quencher group is a BHQ1 quencher group.

[0009] More preferably, a product is prepared for broad-spectrum detection of mycobacteria.

[0010] A kit for broad-spectrum detection of mycobacteria, comprising any one of the above primer-probe combinations.

[0011] Preferably, the above-mentioned kit further includes an internal control probe and an internal control plasmid, the nucleotide sequence of the internal control probe is shown in SEQ ID NO.7, and the nucleotide sequence of the target fragment in the internal control plasmid is shown in SEQ ID NO.8; the 5' end of the internal control probe is coupled with a second fluorescent group, and the 3' end is coupled with a quenching group, and the type of the second fluorescent group is different from that of the first fluorescent group.

[0012] A method for broad-spectrum detection of mycobacteria not for the purpose of disease diagnosis and treatment, using any of the above kits for detection.

[0013] Preferably, the method for broad-spectrum detection of mycobacteria comprises the following steps:

[0014] Choose whether to perform pretreatment according to the type of sample to be tested;

[0015] If sample pretreatment is not required, directly prepare the sample solution for nucleic acid extraction;

[0016] If sample pretreatment is required, the pretreatment process is to add cell lysis buffer (optionally, lysis is generally sufficient for about 30 minutes), and collect the precipitate after centrifugation as the test sample;

[0017] The internal control plasmid is added to the test sample or qPCR reaction solution to extract the nucleic acid in the test sample; the positive control plasmid is added to the internal control plasmid and nuclease-free water and diluted to prepare a positive control;

[0018] Sterile saline was used as the negative control (NCS), and an internal control plasmid (IC) was added to the sample for simultaneous extraction, or the internal control plasmid (IC) was added to the qPCR reaction after nucleic acid extraction. The no-template control (NTC) was nuclease-free water or an internal control plasmid prepared with nuclease-free water.

[0019] Create the first fluorescent group detection channel and the second fluorescent group detection channel on the fluorescent quantitative PCR instrument, perform qPCR reactions on the test sample, no-template control (NTC), negative control (NCS), and positive control (PC), and read the Ct values ​​of the two channels respectively;

[0020] Determine the results based on the Ct value;

[0021] Check the amplification status in the corresponding channels respectively. The threshold line of the test sample amplification curve is 10% of the Ct value of the target channel in the positive control, and the threshold line of the internal control plasmid amplification curve is 10% of the Ct value of the internal control channel in the no-template control or negative control;

[0022] The requirements for quality control results are shown in Table 1 below.

[0023] Table 1 Requirements for quality control results

[0024] The judgment criteria for the sample test results are shown in Table 2 below.

[0025] Table 2 Sample test result judgment criteria

[0026] It should be noted that, in the positive control of the present invention, the type and gene sequence of the positive control plasmid are not limited and can be determined according to the actual situation. Generally speaking, as long as it is a plasmid containing the target sequence for mycobacterium detection, it can be used as the positive control plasmid of the present invention.

[0027] Alternatively, the positive control plasmid can be a mixture of three recombinant plasmids: pUC57-M.tuberculosis, pUC57-M.terrae, and pUC57-M.xenopi.

[0028] It should be noted that if the internal control plasmid is added during the sample nucleic acid extraction process, the Ct value of the second fluorophore channel of the test sample determined to be negative after testing should be within + / - 3 cycles of the Ct value of the second fluorophore channel in the negative extraction control (NCS). If the internal control plasmid is added during the qPCR reaction, the Ct value of the second fluorophore channel of the test sample determined to be negative after testing should be within + / - 2 cycles of the Ct value of the second fluorophore channel in the negative extraction control. If the second fluorophore channel signal is inhibited, retest or appropriate sample treatment should be performed to eliminate the inhibitory factor.

[0029] Compared with the prior art, the primer-probe combination and the corresponding qPCR detection method provided by the present invention are beneficial in that:

[0030] 1. Using the primer-probe combination provided by the present invention for dual fluorescence quantitative PCR, the presence of mycobacterial contamination in the sample can be quickly determined based on the Ct value and amplification curve. More than 160 mycobacterial species can be detected, covering a wide range.

[0031] 2. The detection method provided by the present invention can complete the detection of mycobacterial contamination within a few hours, which greatly shortens the detection time compared with traditional detection methods;

[0032] 3. Strong specificity, with no cross-reaction with human and animal cells, fungi, bacteria other than mycobacteria, mycoplasma, and common gene therapy-related viral genomes;

[0033] 4. The detection sensitivity is high, with the detection limit reaching 100 CFU / ml and the genome detection limit being 10-20 copies / reaction. The sensitivity is not lower than the standard of the Chinese Pharmacopoeia.

[0034] 5. Strong durability and anti-matrix interference ability, introducing 10 7 The mycobacterium can also be detected after the mycobacterium genome is extracted from 100 CFU / ml of 293T / CHO / Vero cells and 1 ml of culture supernatant.

[0035] 6. The present invention sets up an internal control system for the HEX channel (internal control plasmid and internal control probe), which can monitor whether there is inhibition in the extraction of the test sample and the qPCR reaction when detecting mycobacteria, effectively avoiding the occurrence of false negatives, and making the result judgment more accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is the detection capability verification result of the Mycobacterium qPCR detection system in Example 3;

[0037] FIG2 is the sensitivity verification result of the standard plasmid in Example 3;

[0038] FIG3 is a cross-reaction verification result of the primer-probe combination of the present invention with non-mycobacteria in Example 4;

[0039] FIG4 shows the results of the robustness verification of the mycobacterium detection method in Example 5;

[0040] FIG5 shows the results of the robustness verification of the mycobacterium detection method in Example 5;

[0041] FIG6 is the specificity verification result of the internal control probe in Example 6;

[0042] FIG7 is a result of verifying whether there is cross-reaction between mycobacteria detection and internal control plasmid in Example 6;

[0043] FIG8 shows the detection results of Example 7 using the primer-probe combination and detection method of the present invention. DETAILED DESCRIPTION

[0044] TaqMan PCR technology is a type of real-time fluorescence PCR. Compared to traditional PCR, it adds a probe labeled with a fluorescent marker and a quencher at either end to the reaction system. When the probe structure is intact, the fluorescence energy emitted by the fluorescent marker is transferred to the quencher, resulting in a quenching effect. If the target sequence is present during amplification, the probe molecule is gradually hydrolyzed and cleaved, and the fluorescent reporter and quencher groups dissociate from each other, blocking the fluorescence resonance energy transfer effect between them. The fluorescent reporter group then emits a fluorescent signal. As amplification proceeds, the fluorescence signal increases linearly with the amplification of the target fragment.

[0045] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0046] In the primer-probe combinations used in the following examples, the detection probe is coupled to a first fluorescent group at its 5' end and a quencher at its 3' end; the internal control probe is coupled to a second fluorescent group at its 5' end and a quencher at its 3' end.

[0047] Optionally, the first fluorescent group can be selected from FAM, TET, NED, ROX, CY3, CY5, VIC, JOE, HEX fluorescent groups, or other fluorescent groups.

[0048] Optionally, the second fluorescent group can also be selected from FAM, TET, NED, ROX, CY3, CY5, VIC, JOE, HEX fluorescent groups, or other fluorescent groups, but the second fluorescent group cannot be the same as the first fluorescent group.

[0049] Optionally, the quenching group can be selected from TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1, BHQ2 quenching groups, or other quenching groups.

[0050] There is no requirement for the selection of quenching groups on the detection probe and the internal control probe, and they can be the same or different.

[0051] In the following embodiments, the first fluorescent group is selected from the FAM fluorescent group, the second fluorescent group is selected from the HEX fluorescent group, and the quenching group is selected from the BHQ1 fluorescent group.

[0052] The detection primers used in the following examples were all synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd., and the detection probe, internal control probe and internal control plasmid were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0053] The RNase-free water used in the following examples is also Nuclease-free water, that is, it does not contain RNase or DNase.

[0054] Unless otherwise specified, the technical solutions used in the following examples all employ conventional molecular biological techniques. The human cells (e.g., human embryonic kidney cells 293 and 293T, human umbilical cord mesenchymal stem cells MSC), monkey cell lines (e.g., African green monkey kidney cells Vero), hamster cell lines (e.g., hamster kidney cells BHK-21, hamster ovary cells CHO-K1), bovine cell lines (e.g., bovine kidney cells MDBK), and porcine cell lines (e.g., porcine kidney cells PK-15) used are all independently preserved by the company. The adenovirus HAd-5, adeno-associated virus AAV-2, Escherichia coli, Clostridium sporogenes, Staphylococcus aureus, Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Bacillus cereus, Micrococcus luteus, Corynebacterium diphtheria, Mycoplasma pneumoniae, and Mycoplasma oral used are all independently preserved by the company.

[0055] Alternatively, when adding the internal control plasmid to the test sample, the internal control plasmid is present at 700 copies / sample. When adding the internal control plasmid to the qPCR reaction solution, the internal control plasmid is present at 200 copies / well.

[0056] Optionally, in each reaction well, the positive control plasmid and the internal control plasmid in the positive control are 1000 copies / well and 200 copies / well, respectively.

[0057] Unless otherwise specified, the reagents (e.g., qPCR reaction solutions, reagents of the nucleic acid extraction system, buffers, etc.), kits, and technical solutions used in the following examples were all commercially available reagents and kits, as well as conventional molecular biology techniques in the industry.

[0058] Example 1: Synthesis of primer probes

[0059] 1. Mycobacterium primer and probe design:

[0060] Common pathogenic mycobacterial species were identified by consulting the European Pharmacopoeia, the Chinese Pharmacopoeia, the Guidelines for the Diagnosis and Treatment of Nontuberculous Mycobacterial Diseases (2020 Edition), and related literature. Their 16S rRNA sequences were downloaded from NCBI. Mycobacterial 16S rRNA sequences were aligned with those of other bacteria, mycoplasmas, and fungi. Primers and probes specifically for mycobacterial detection were designed. These primers consisted of two forward primers, one reverse primer, and three detection probes. The primers were synthesized by Tianyi Huiyuan, and the detection probes were synthesized by Sangon. The sequences are shown in Table 3 below.

[0061] Table 3 Nucleotide sequences of primer-probe combinations

[0062] 2. Internal control plasmid and probe sequence

[0063] The target fragment of the internal control plasmid was artificially synthesized and the recombinant plasmid pUC57-IC was constructed. This plasmid was used as a template and added to the Mycobacterium qPCR detection system. The nucleotide sequence of the target fragment is shown in SEQ ID NO. 8, specifically:

[0064] The nucleotide sequence of the corresponding internal control probe ProbeIC is shown in SEQ ID NO.7, specifically:

[0065] HEX-catgcgagactgacctgagctaggagctg-BHQ1

[0066] Example 2: Establishment of Mycobacterium qPCR Detection System and Addition of Internal Control Plasmid

[0067] Synthesize the pUC57-IC recombinant plasmid and determine its concentration. Calculate the pUC57-IC plasmid copy number according to the formula and perform gradient dilution to 1×10 3 Keep copies / μl for future use.

[0068] Synthesize the pUC57-M.xenopi recombinant plasmid, which shares primers with the internal control, and measure its concentration. Calculate the plasmid copy number using the formula and serially dilute to 10 copies / μl. Use TaKaRa's Probe qPCR Mix and prepare the qPCR reaction system according to Table 4, ensuring 100 copies of pUC57-M.xenopi per reaction well. Perform dual-channel qPCR according to the protocol in Table 5.

[0069] Table 4 qPCR detection reaction system

[0070] Table 5 qPCR program

[0071] It has been confirmed that adding 200 copies / reaction of IC does not affect the detection sensitivity Ct value of pUC57-M.xenopi, and the internal control channel can stably detect.

[0072] Example 3: Verification of the detection capability of the Mycobacterium qPCR detection system

[0073] 1. Prediction of Mycobacterium Detection Range

[0074] The designed primer probes were subjected to specific sequence search and comparison using Primer-BLAST in NCBI. The results showed that the primer probes in the present invention can cover 167 species of mycobacteria (including 39 pathogenic mycobacteria). The statistical results are shown in Table 6.

[0075] Table 6 Statistical results of specific sequence search and alignment

[0076] 2. Verification of Mycobacterium Detection Range

[0077] Eleven mycobacterial species (Mycobacterium avium, M. terrae, M. shimoidei, M. kansasii, M. asiaticum, M. scrofulaceum, M. gordonae, M. abscessus, M. fortuitum, M. phlei, and M. tuberculosis) listed in the national reference materials in the Mycobacterium tuberculosis PCR detection kit of the China Food and Drug Inspection Institutes were used as materials. The genomes of the 11 mycobacterial reference materials were extracted and detected using the qPCR detection reaction system and qPCR procedure shown in Tables 4 and 5 above.

[0078] As shown in FIG1 , 11 mycobacteria were detected 100% of the time, and the amplification results are summarized in Table 7 below.

[0079] Table 7 Summary of amplification

[0080] 3. Standard plasmid sensitivity verification and standard curve establishment

[0081] Six mycobacterial species (M. tuberculosis, M. lepare, M. scrofulaceum, M. xenopi, M. terrae, and M. phlei) were selected to construct the mycobacterial 16S rRNA standard plasmid. The mycobacterial 16S rRNA standard plasmid was extracted using the Plasmid Mini Kit I (OMEGA). The plasmid concentration was measured, the plasmid copy number was calculated, and the plasmid was diluted to 2 × 10 6 -2×10 2 The standard curve was made by taking 2×10 1 copies / μl plasmid standard solution was used as a plasmid sensitivity control.

[0082] qPCR was performed using the amplification system and reaction procedures in Tables 4 and 5 above. The experiment was repeated three times on three different days, and the plasmid sensitivity control was repeated in 8 wells each time.

[0083] The test results showed that the amplification efficiency of the six mycobacteria was >90%, and the correlation coefficient R 2 The sensitivity of the plasmids reached 100 copies / reaction. The experimental results are shown in Table 8 and Figure 2.

[0084] Table 8 Standard plasmid sensitivity verification results

[0085] 4. Mycobacterial genome detection limit

[0086] Using three mycobacteria, M. bovis BCG (Shanghai Ruichu Biotechnology Co., Ltd., BCG lyophilized powder), M. phlei (plate bacteria obtained by our own culture method), and M. gordonae (purchased from ATCC) as examples, genomic DNA was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (TaKaRa, 9763). The copy number was calculated based on the genome size and the measured concentration, and a serial dilution was performed. 100, 50, 20, and 10 copies / reaction were used as reaction samples, respectively. Three independent replicates were performed on three different days, with 8 replicates per well for each dilution in each experiment. The reaction system and reaction procedure are the same as those in Tables 4 and 5.

[0087] The results are shown in Table 9 below. The detection limit of M. bovis BCG genome nucleic acid is 20 copies / reaction; the detection limit of M. phlei genome is 10 copies / reaction; and the detection limit of M. gordonae genome is 20 copies / reaction.

[0088] Table 9 Detection limit verification results

[0089] 5. Verification of the detection limit of mycobacteria standards

[0090] The Mycobacterium tuberculosis PCR detection kit of China Food and Drug Inspection Institute was used with the precision reference material (Mycobacterium tuberculosis CMCC 93009, 1×10 2 bacteria / ml) and Mycobacterium smegmatis standard (0.5-1×10 3 CFU / ml, diluted 10-fold before use) was used as the material. Using our company's developed mycobacterial genome extraction method, samples were extracted four times over four days, with two replicates each time. qPCR analysis was performed daily with three replicates per sample, for a total of 24 wells across four experiments. The reaction system and procedure are shown in Tables 4 and 5.

[0091] The results are shown in Table 10: The qPCR detection method in this protocol can stably detect Mycobacterium tuberculosis (100 bacteria / ml) and Mycobacterium smegmatis (100 CFU / ml), which meets the sensitivity requirements for mycobacterium detection in the current edition of the Chinese Pharmacopoeia.

[0092] Table 10 Verification results of detection limit of mycobacteria standards

[0093] Example 4: Specificity Verification of Mycobacterium Detection Method

[0094] 1. Extraction of genomic DNA from various biological cells

[0095] (1) Human and animal cell genomic DNA

[0096] Porcine kidney cells PK-15, bovine kidney cells MDBK, African green monkey kidney cells Vero, Chinese hamster ovary cells CHO-K1, human embryonic kidney cells 293 and 293T, hamster kidney cells BHK-21, and human umbilical cord mesenchymal stem cells MSC are all preserved by our company and used Genomic DNA was extracted using Mini kit (50) (QIAGEN, 51304).

[0097] (2) Viral genomic DNA

[0098] Adenovirus HAd-5 and adeno-associated virus AAV-2 were stored by our company, and genomic DNA was extracted using the Viral Nucleic Acid purification kit (simgen, 4002050).

[0099] (3) Bacterial genomic DNA

[0100] Escherichia coli, Clostridium sporogenes, Staphylococcus aureus, Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Bacillus cereus, Micrococcus luteus, and Corynebacterium diphtheria were all maintained by our company, and genomic DNA was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0 (TaKaRa, 9763).

[0101] (4) Common Mycoplasma genomic DNA

[0102] Mycoplasma pneumoniae and Mycoplasma oral were stored in our company, and genomic DNA was extracted using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (TaKaRa, 9763).

[0103] 2. Preparation of working concentration genomic DNA

[0104] The concentrations of the above-mentioned cell, bacterial, and mycoplasma genomic DNA were measured using a micro-spectrophotometer. Based on the measured concentrations, the bacterial and mycoplasma genomes were diluted to 0.2 ng / μl with RNase-free water, the human and animal cell genomes were diluted to 20 ng / μl, and the adenovirus HAd-5 and adeno-associated virus AAV-2 were taken in 1 ml with a physical titer of 10 8 After DNA was extracted from the virus solution containing 5 copies / ml, 5 μl was used as a template for qPCR. The pUC57-M. tuberculosis recombinant plasmid was used as a positive control plasmid.

[0105] The qPCR reaction system and amplification procedure were the same as those in Tables 4 and 5 above.

[0106] The results are shown in FIG3 . It can be seen that the primer-probe combination of the present invention has no cross-reaction with non-mycobacteria. The amplification of non-mycobacterial genomic DNA is summarized in Table 11 .

[0107] Table 11 Common non-mycobacterial cells

[0108] Example 5: Verification of the robustness of the mycobacterium detection method

[0109] Mycobacterium is an intracellular bacterium. When testing cell samples contaminated with mycobacteria, the culture supernatant and cell lysate need to be co-extracted and tested. The cellular DNA introduced during the extraction process may inhibit the amplification of mycobacterial targets, thereby affecting the detection. Therefore, durability verification is required.

[0110] The 293T / CHO / Vero cell genome was extracted, the concentration was measured, and diluted to 80 ng / μl for later use. Plasmid standards of M. terrae, M. xenopi, and M. tuberculosis at 100 copies / reaction were used as controls. 400 ng / reaction of the 293T / CHO / Vero cell genome was introduced into 100 copies / reaction of the M. terrae, M. xenopi, and M. tuberculosis plasmid standards, respectively. All of these samples were subjected to mycobacterial qPCR detection. The qPCR reaction system and procedure were the same as in Tables 4 and 5.

[0111] The validation results are shown in FIG4 and Table 12. Compared with the control without 293T / CHO / Vero cell genome, the introduction of 293T / CHO / Vero cell genome did not lead to false negative results in mycobacterium detection.

[0112] Table 12 Durability validation results of the Mycobacterium detection method

[0113] The M. tuberculosis precision reference material (100 bacteria) in the M. tuberculosis PCR detection kit of the China Food and Drug Inspection Institute and the M. smegmatis (Mycobacterium smegmatis) standard material (100 CFU) of Beijing Sanyao Technology Development Co., Ltd. were used as materials. 10 7 293T / CHO / Vero cell samples were prepared. Mycobacterium genomic nucleic acid was extracted using our company's developed mycobacterium genome extraction method and then tested for mycobacteria using qPCR. The qPCR reaction system and procedure were the same as shown in Tables 4 and 5.

[0114] The verification results are shown in Figure 5 and Table 13. No amplification signals were found in the cell samples without the addition of mycobacteria, while samples artificially contaminated with tuberculosis and Mycobacterium smegmatis tested positive.

[0115] Table 13 Durability validation results of the Mycobacterium detection method

[0116] Example 6: Verification of internal control probe specificity, mycobacterium qPCR detection, and cross-reaction with internal control plasmid

[0117] 1. Specificity verification of internal control probe

[0118] Genomes of 11 mycobacteria, 8 human and animal cells, 2 viruses, 9 bacteria, and 2 mycoplasmas were extracted and used for validation using the mycobacterium qPCR detection method of this protocol. The qPCR reaction system and reaction procedure are the same as those shown in Tables 4 and 5 above.

[0119] The experimental results are shown in Table 14 and Figure 6, indicating that the internal control probe has no cross-reaction with human and animal cells, viruses, mycobacteria, bacteria, and mycoplasma genomes.

[0120] Table 14 Cross-reaction verification results

[0121] 2. Verification of cross-reaction between mycobacterium qPCR detection and internal control plasmid

[0122] During the extraction of the mycobacterial genome, 1 ml of sterile saline was taken as a negative control NCS, and 700 copies / sample of the internal control plasmid were introduced. After the extraction, mycobacterial qPCR detection was performed. The reaction system and reaction procedure were the same as those in Tables 4 and 5 above.

[0123] As shown in Figure 7 , the HEX channel of the negative control sample NCS produced a normal amplification curve, while the FAM channel did not produce a fluorescent signal, proving that the introduced internal control plasmid had no cross-reaction with the mycobacterium detection, and that the introduction of the internal control plasmid would not lead to false positives during the sample detection process.

[0124] Example 7: Application example of mycobacterium detection method

[0125] The qPCR method in this protocol was used to test the CHO cell seed bank and working bank samples for mycobacteria to determine whether there was mycobacterial contamination. Internal control plasmids were added during the extraction process, and DNA extraction of test samples and negative controls was performed using MinElute Virus Spin Kit (QIAGEN 57704) can be operated according to the product instructions. The specific method is as follows:

[0126] Negative control: Take 1 ml of sterile saline, add 700 copies of internal control plasmid after pretreatment, and extract DNA;

[0127] Test samples: Take 10 7 CHO cells from a seed bank or working bank and 1 ml of culture supernatant were pretreated and 700 copies of the internal control plasmid were added to extract DNA.

[0128] Positive control: dilute the positive control plasmid (pUC57-M.tuberculosis, pUC57-M.terrae, pUC57-M.xenopi recombinant plasmid mixture) in the kit to 100 copies / μl, and add 10 μl to the qPCR reaction tube. Dilute the internal control plasmid in the kit to 1000 copies / μl, and add 0.2 μl to the qPCR reaction tube.

[0129] Specifically, the pUC57-M.tuberculosis, pUC57-M.terrae, and pUC57-M.xenopi recombinant plasmids were obtained by transferring the 16S rRNA nucleic acid sequences of Mycobacterium tuberculosis, Mycobacterium terrae, and Mycobacterium xenopi, which can monitor the qPCR reaction of mycobacteria, into the pUC57 plasmid and extracting them using the Plasmid Mini Kit I (OMEGA). After measuring the concentration and calculating the plasmid copy number, the plasmids were diluted to 1×10 8 copies / μl, and mix the three in equal volumes for later use.

[0130] No-template control: Add RNase-free water to the qPCR reaction tube.

[0131] The qPCR reaction system and reaction procedures were the same as those in Tables 4 and 5 above.

[0132] The test results are shown in Figure 8 and Table 15. Based on the quality control result requirements in Table 1 and the sample test result judgment criteria in Table 2, it was determined that there was no mycobacterium contamination in the CHO cell seed bank and working bank.

[0133] Table 15 Test results

[0134] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A primer-probe combination for broad-spectrum detection of mycobacteria, characterized in that: It comprises a forward primer, a reverse primer and a probe, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3, and the nucleotide sequence of the detection probe is shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; the 5' end of the detection probe is coupled with a first fluorescent group, and the 3' end is coupled with a quenching group.

2. The primer-probe combination for broad-spectrum detection of mycobacteria according to claim 1, characterized in that: The first fluorescent group is a FAM, TET, NED, ROX, CY3, CY5, VIC, JOE or HEX fluorescent group; the quenching group is a TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1 or BHQ2 quenching group.

3. The primer-probe combination for broad-spectrum detection of mycobacteria according to claim 1, characterized in that: The first fluorescent group is a FAM fluorescent group, and the quenching group is a BHQ1 quenching group.

4. Use of the primer-probe combination according to any one of claims 1 to 3, characterized in that: Prepare products for broad-spectrum detection of mycobacteria.

5. A kit for broad-spectrum detection of mycobacteria, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 3.

6. The kit for broad-spectrum detection of mycobacteria according to claim 5, characterized in that: It also includes an internal control probe and an internal control plasmid, the nucleotide sequence of the internal control probe is shown in SEQ ID NO.7, and the nucleotide sequence of the target fragment in the internal control plasmid is shown in SEQ ID NO.8; the 5' end of the internal control probe is coupled with a second fluorescent group, and the 3' end is coupled with a quenching group, and the type of the second fluorescent group is different from that of the first fluorescent group.

7. A method for broad-spectrum detection of mycobacteria not for the purpose of disease diagnosis and treatment, characterized in that: The test kit according to claim 5 or 6 is used for detection.

8. The method according to claim 7, characterized in that The method includes the steps of extracting nucleic acid from the test sample, specifically: Choose whether to perform a pretreatment process according to the type of sample to be tested; If sample pretreatment is not required, the sample solution is directly prepared for nucleic acid extraction; if sample pretreatment is required, the pretreatment process is to add cell lysis solution, and collect the precipitate after centrifugation as the sample; Add the internal control plasmid to the test sample or qPCR reaction solution to extract the nucleic acid in the test sample; add the positive control plasmid to the internal control plasmid and nuclease-free water and dilute to prepare a positive control; Sterile physiological saline was used as a negative control, and an internal control plasmid was added to extract nucleic acid simultaneously with the test sample, or the internal control plasmid was added to the qPCR reaction after the nucleic acid was extracted; the no-template control was nuclease-free water, or an internal control plasmid prepared with nuclease-free water; Creating a first fluorescent group detection channel and a second fluorescent group detection channel on a fluorescent quantitative PCR instrument, performing qPCR reactions on the test sample, the no-template control, the negative control, and the positive control, respectively, and reading the Ct values ​​of the two channels respectively; determining the quality control results of the positive control, the negative control, and the no-template control, as well as the detection result of the test sample based on the Ct value; When the Ct of the first fluorescent group detection channel of the test sample is <40 and there is a normal amplification curve, the mycobacterium detection result is determined to be positive; When the first fluorescent group detection channel of the test sample has no Ct value and no normal amplification curve, if the internal control plasmid is added during the nucleic acid extraction process, the second fluorescent group detection channel has a normal amplification curve and the Ct value of the second fluorescent group detection channel in the negative control does not differ by more than 3 cycles; if the internal control plasmid is added during the qPCR reaction process, the second fluorescent group detection channel has a normal amplification curve and the Ct value of the second fluorescent group detection channel in the negative control does not differ by more than 2 cycles, then the mycobacterium detection result is finally determined to be negative; When both the first fluorescent group detection channel and the second fluorescent group detection channel of the test product have no Ct value and no normal amplification curve; or when the first fluorescent group detection channel has a Ct ≥ 40 and a normal amplification curve, and the second fluorescent group detection channel has no Ct value and no normal amplification curve, it is determined that the qPCR reaction is inhibited; If the Ct of the first fluorescent group detection channel of the test sample is ≥40 and has a normal amplification curve, and the Ct of the second fluorescent group detection channel is <40 and has a normal amplification curve, the result is judged to be invalid.

9. The method according to claim 8, characterized in that The criteria for quality control results of positive control, negative control and no-template control are as follows: Check the amplification status in the corresponding channels respectively. The threshold line of the test sample detection amplification curve is 10% of the Ct value of the target channel in the positive control, and the threshold line of the internal control plasmid amplification curve is 10% of the Ct value of the internal control channel in the no-template control or negative control; Requirements for quality control results: The first fluorescent group detection channel and the second fluorescent group detection channel of the positive control are both positive; the first fluorescent group detection channel of the negative control is negative, and the second fluorescent group detection channel is positive; the first fluorescent group detection channel of the no-template control is negative, the second fluorescent group detection channel is positive when the internal control plasmid is added, and the second fluorescent group detection channel is negative when the internal control plasmid is not added.

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