Primer group, composition, kit and device for detecting corynebacterium murine

Abstract

The invention relates to a primer group, a composition, a kit and a device for detecting corynebacterium murine. The composition comprises an upstream primer and a downstream primer of target double-stranded DNA, a TaqMan fluorescent probe, recombinase, T7RNA polymerase, transcriptase, ATP energy regeneration enzyme, melting enzyme, single-stranded binding protein and cutting enzyme. According to the invention, nucleic acid amplification enzyme and signal amplification enzyme are integrated together, the purpose of amplifying target double-stranded DNA to 109 times within 10-30 minutes under the condition of constant temperature of 25 DEG C is realized, and each nucleic acid amplification product can generate more than 10,000 fluorescence signals under the condition of constant temperature of 25 DEG C, so that efficient and rapid detection of samples is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial detection, and in particular to a primer set, a composition, a kit and a device for detecting Corynebacterium muridarum. Background Art

[0002] Corynebacterium murium is a common pathogen in experimental rodents such as mice, rats, and hamsters, which can cause illness or even death, affecting the accuracy and reliability of experimental results. Natural infection is mostly latent. When animals are exposed to stress factors such as strong radiation, surgical treatment, transportation, malnutrition, or receive immunosuppressants, radiation, and lack of pantothenate in food, latent infection will become explicit, causing acute disease, causing animals to become emaciated, have ruffled fur, have difficulty breathing, swollen joints, skin ulcers and abscesses, and even form subcutaneous fistulas. Limbs may also fall off due to necrosis, and the morbidity and mortality rates are both high.

[0003] According to the national standard for experimental animals in my country (GB 14922-2022 Laboratory Animal Microorganisms, Parasites Grade and Monitoring), it is clearly stipulated that specific pathogen-free (SPF) experimental rats and mice must exclude Corynebacterium muris. In recent years, the results of laboratory animal quality monitoring at home and abroad also show that the positive rate of Corynebacterium muris in experimental rats and mice is relatively high, which will affect the quality of experimental animals and interfere with the experimental results. According to the current national standard (GB / T GBT 14926.9-2001 Laboratory Animal Corynebacterium muris Detection Method), the detection of Corynebacterium muris requires the isolation, culture and biochemical identification of samples. This method takes at least 3 days, the detection cycle is long, the operation is cumbersome, time-consuming and laborious. If the DNA amplification method is used, although Corynebacterium muris can be detected in a relatively fast time, it still faces many problems.

[0004] At present, the methods of DNA amplification include conventional PCR and isothermal PCR. These technologies can quickly amplify the target DNA by 1 million times or even higher in a short time. Since its introduction in 1983, conventional PCR has relied on three-temperature (melting, annealing, extension) cycles, and has very high requirements for the stability of PCR instruments. Although two-temperature cycles can shorten the reaction time, the activity of DNA polymerase is not at the optimal temperature, so only shorter fragments can be amplified, and the time consumption has not been greatly reduced. Isothermal PCR relies on a stable temperature higher than room temperature (for example, 60°C), and still requires an instrument with precise temperature control. General room-temperature PCR relies on recombinase and polymerase with chain displacement to amplify shorter sequences, requiring an ATP regeneration system, which makes the reaction system more complicated and easily induces side reactions, interfering with the detection effect. Since the performance and operation mode of molecular diagnostic reagents are determined by their core enzyme components, new room-temperature nucleic acid amplification detection can be performed based on enzyme-mediated dual exponential amplification technology.

[0005] In the detection of experimental animals, the most common practice is to detect Corynebacterium spp. in experimental animals through isolation, culture and biochemical identification of bacteria. Although this method is the gold standard for the detection of pathogenic microorganisms, its detection cycle is long and the operation is cumbersome. At the same time, the professional level of the tester has a great influence on the results of biochemical culture.

[0006] Compared with traditional biochemical testing, PCR testing by amplifying the DNA of Corynebacterium muridarum is not only highly sensitive but also can help testing agencies determine whether Corynebacterium muridarum is present in samples in a relatively short period of time. For experimental animal pathogens, the common nucleic acid detection methods currently include ordinary PCR detection and constant temperature PCR detection. With the improvement of laboratory hardware conditions and the professional level of personnel, in addition to PCR detection methods, some group standards have begun to use constant temperature detection methods such as loop-mediated isothermal amplification (LAMP) and reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to detect some experimental animal pathogens.

[0007] Although the common / constant temperature PCR detection methods have shortened the detection time of pathogenic microorganisms in experimental animals to a certain extent, they still face the problems of large investment in laboratory hardware and the need for professional personnel to operate, and there is still room for improvement in the detection time. These all require experimental animal institutions to invest a large amount of costs, among which hardware conditions and environmental requirements directly affect the judgment of the results. Providing an on-site molecular rapid detection technology and applying it to scenarios such as experimental animal epidemic prevention is the most effective way to fundamentally solve the source of Corynebacterium muridarum. In general, there is an urgent need to provide an on-site instant detection kit and method for Corynebacterium muridarum with accuracy comparable to that of professional laboratories and with the advantages of fast detection time, simple operation, and compactness and portability. Summary of the invention

[0008] In view of the problems in the prior art that the detection of Corynebacterium muridarum takes a long time and requires professional equipment and professional personnel to operate, the present invention provides a primer set, a composition, a kit, and a device for detecting Corynebacterium muridarum. The present invention can carry out detection at any location, has a simple operation process, is suitable for use by anyone, and can detect Corynebacterium muridarum in a sample within 30 minutes at 25°C.

[0009] The technical solution provided by the present invention is specifically as follows:

[0010] In a first aspect, the present invention provides a primer set for RAA amplification of Corynebacterium muridarum nucleic acid, comprising an upstream primer and a downstream primer of a target double-stranded DNA;

[0011] The nucleotide sequence of the target double-stranded DNA is shown in SEQ ID NO.19;

[0012] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5;

[0013] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.:7.

[0014] In a second aspect, the present invention provides a composition for detecting Corynebacterium avium, comprising:

[0015] An upstream primer and a downstream primer of the target double-stranded DNA, wherein the upstream primer comprises a T7 promoter;

[0016] A recombinase that can catalyze the formation of a double-stranded DNA containing a T7 promoter by an upstream primer and a first single-stranded target double-stranded DNA;

[0017] T7 RNA polymerase, which is used to catalyze the transcription of double-stranded DNA containing a T7 promoter to generate single-stranded RNA;

[0018] Transcriptase, which catalyzes the reverse transcription of single-stranded RNA into double-stranded DNA / RNA;

[0019] ATP energy regenerating enzyme, which is used to provide ATP energy in DNA recombination and transcription processes;

[0020] Helicase, which is used to catalyze the hydrolysis of DNA / RNA double strands into RNA single strands and DNA single strands;

[0021] Single-strand binding protein, which is used to bind to single-stranded DNA and prevent the self-polymerization of single-stranded DNA;

[0022] TaqMan fluorescent probe, which includes an RNA chain that can bind to a single-stranded DNA, and the 5' end and 3' end of the RNA chain have a reporter group and a quencher group respectively;

[0023] A cleavage enzyme, which is used to cleave the RNA chain of the TaqMan fluorescent probe to release the fluorescent group when the TaqMan fluorescent probe is bound to the DNA single chain;

[0024] in:

[0025] The nucleotide sequence of the target double-stranded DNA is shown in SEQ ID NO.19;

[0026] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5;

[0027] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.: 7;

[0028] The nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.:18.

[0029] In some embodiments, the quencher group of the TaqMan fluorescent probe is one of BHQ, BHQ1, BHQ2, and TAMRA, and the reporter group is one of FAM, FAM-X, CY3, CY5, HEX, and TET.

[0030] In some embodiments, the Corynebacterium muridarum detection composition further comprises a plasmid standard of the target double-stranded DNA.

[0031] In some embodiments, the Corynebacterium muridarum detection composition is used to amplify and detect target double-stranded DNA at a constant temperature of 25°C.

[0032] In some embodiments, the Corynebacterium muridarum detection composition provided by the present invention further comprises a plasmid standard of the Corynebacterium muridarum 16SrRNA gene fragment.

[0033] In a third aspect, the present invention provides a Corynebacterium muridarum detection kit based on enzyme-mediated room-temperature nucleic acid amplification, wherein the Corynebacterium muridarum detection kit comprises the above-mentioned Corynebacterium muridarum detection composition.

[0034] In some embodiments, the Corynebacterium muridarum detection kit provided by the present invention further comprises a lysis solution, wherein the lysis solution comprises a broad-spectrum protease and a physiological buffer.

[0035] In some embodiments, the Corynebacterium muridarum detection kit provided by the present invention comprises a lyophilization protectant, and each component in the Corynebacterium muridarum detection composition is packaged in the form of lyophilized powder.

[0036] In a fourth aspect, the present invention provides a device for detecting Corynebacterium avium, comprising:

[0037] A DNA extraction module, which is used to extract sample DNA;

[0038] An amplification module, which is used to amplify the sample DNA using the above-mentioned Corynebacterium muridarum detection composition to obtain an amplification product;

[0039] The fluorescence detection module is used to detect the fluorescence signal of the amplification product.

[0040] The Corynebacterium muridarum detection device also includes a judgment module:

[0041] The judgment module judges whether the sample contains Corynebacterium ratum according to the fluorescent signal; if there is no fluorescent signal, it is judged that the sample does not contain Corynebacterium ratum; if there is a fluorescent signal, it is judged that the sample contains Corynebacterium ratum.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The DNA extraction process of the present invention is simplified, and the DNA extraction work can be completed by only adding a lysis solution and performing a 10-minute heat treatment.

[0044] 2. The present invention integrates a nucleic acid amplification enzyme group to achieve the amplification of the target double-stranded DNA to 10 within 10 to 30 minutes at a constant temperature of 25°C. 9 At the same time, by integrating the signal amplification enzyme system, each nucleic acid amplification product can generate more than 10,000 fluorescent signals at a constant temperature of 25°C, thereby achieving efficient and rapid detection of samples.

[0045] 3. The molecular detection of Corynebacterium muridarum in the present invention has extremely high specificity and a sensitivity of up to 1 copy / μL, which is helpful for early detection of samples infected with Corynebacterium muridarum.

[0046] 4. The present invention can be used in conjunction with a portable metal bath and a portable fluorescence detector, and can be carried to any location such as a rural site for detection, meeting the needs of various application scenarios such as laboratory environment detection and animal cage detection.

[0047] 5. The operation process of the present invention is simple, suitable for use by all groups of people, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The schematic diagram is a schematic diagram of the present invention for detecting Corynebacterium muridarum based on enzyme-mediated nucleic acid amplification at room temperature.

[0049] Figure 2-5 The TaqMan fluorescent probe screening results of Example 1 of the present invention;

[0050] Figure 6 The results of DNA downstream primer screening in Example 2 of the present invention are as follows;

[0051] Figure 7The results of DNA upstream primer screening in Example 3 of the present invention are as follows;

[0052] Figure 8 The specific experimental results of Example 4 of the present invention;

[0053] Figure 9-10 The sensitivity test results of Example 5 of the present invention are shown below:

[0054] Figure 11-12 The test results of the experimental example of the present invention are shown in FIG. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0056] The primer set for RAA amplification of coryneform bacteria nucleic acid provided by the present invention comprises an upstream primer and a downstream primer of a target double-stranded DNA; the nucleotide sequence of the target double-stranded DNA is shown in SEQ ID NO.19; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.:5; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.:7.

[0057] The present invention designs DNA upstream primers and DNA downstream primers for the conserved region of 16S rRNA sequence in the genome of Corynebacterium rapae (hereinafter referred to as target double-stranded DNA), and uses them to perform room-temperature nucleic acid amplification on DNA extracted from the sample to be tested, and can obtain 10 within 10 to 30 minutes under a constant temperature of 25°C. 9 times the nucleic acid amplification product.

[0058] The Corynebacterium avium detection composition provided by the present invention comprises:

[0059] An upstream primer and a downstream primer of the target double-stranded DNA, wherein the upstream primer comprises a T7 promoter;

[0060] A recombinase that can catalyze the formation of a double-stranded DNA containing a T7 promoter by an upstream primer and a first single-stranded target double-stranded DNA;

[0061] T7 RNA polymerase, which is used to catalyze the transcription of double-stranded DNA containing a T7 promoter to generate single-stranded RNA;

[0062] Transcriptase, which catalyzes the reverse transcription of single-stranded RNA into double-stranded DNA / RNA;

[0063] ATP energy regenerating enzyme, which is used to provide ATP energy in DNA recombination and transcription processes;

[0064] Helicase, which is used to catalyze the hydrolysis of DNA / RNA double strands into RNA single strands and DNA single strands;

[0065] Single-strand binding protein, which is used to bind to single-stranded DNA and prevent the self-polymerization of single-stranded DNA;

[0066] TaqMan fluorescent probe, which includes an RNA chain that can bind to a single-stranded DNA, and the 5' end and 3' end of the RNA chain have a reporter group and a quencher group respectively;

[0067] A cleavage enzyme, which is used to cleave the RNA chain of the TaqMan fluorescent probe to release the fluorescent group when the TaqMan fluorescent probe is bound to the DNA single chain;

[0068] in:

[0069] The nucleotide sequence of the target double-stranded DNA is shown in SEQ ID NO.19;

[0070] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5;

[0071] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.: 7;

[0072] The nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.:18.

[0073] Simply put, the composition includes a nucleic acid amplification enzyme group and a signal amplification enzyme group. The nucleic acid amplification enzyme group contains 4 enzymes (recombinase, single-stranded binding protein, polymerase, ATP energy regeneration enzyme), and the signal amplification enzyme group contains 2 enzymes (transcriptase and cleavage enzyme). The present invention integrates the two groups of proteins together, so that nucleic acid amplification and signal amplification can be carried out in one reaction tube, simplifying the operation.

[0074] The present invention uses the above-mentioned Corynebacterium avium detection composition to achieve 10% detection within 10 to 30 minutes under a constant temperature environment of 25°C. 9 The signal amplifier enzyme group can enable each nucleic acid amplification product to generate more than 10,000 fluorescent signals, significantly improving the speed and sensitivity of detection.

[0075] The TaqMan fluorescent probe used in the present invention is used to display the fluorescent signal generated during the target double-stranded DNA amplification process, and the fluorescent signal is proportional to the total amount of DNA bound to the TaqMan fluorescent probe. In some embodiments of the present invention, the quenching group of the TaqMan fluorescent probe is one of BHQ, BHQ1, BHQ2, and TAMRA, and the reporter group is one of FAM, FAM-X, CY3, CY5, HEX, and TET. Preferably, the 5' end of the TaqMan fluorescent probe is FAM and the 3' end is BHQ1.

[0076] In some embodiments, the Corynebacterium muridarum detection composition further comprises a plasmid standard of the target double-stranded DNA for establishing a standard curve, or for calibrating and verifying the accuracy of quantitative detection of the target double-stranded DNA using the Corynebacterium muridarum detection composition.

[0077] In some embodiments, the Corynebacterium muridarum detection composition is used to amplify and detect target double-stranded DNA at 25° C. without the need for heating, and is suitable for scenarios where heating is inconvenient, and has broad application prospects.

[0078] The Corynebacterium muridarum detection kit provided by the present invention comprises the Corynebacterium muridarum detection composition, which comprises a primer pair and a TaqMan fluorescent probe for optimizing the RAA reaction. The use of the kit simplifies the detection process of Corynebacterium muridarum and improves the sensitivity and specificity of the detection. Through the pre-configured reagent combination, the user can quickly start the Corynebacterium muridarum detection without the need to prepare a reaction mixture separately, thereby reducing the complexity of the experimental operation and the potential risk of contamination.

[0079] In some embodiments, the mouse coryneformis detection kit provided by the present invention further comprises a lysate, which is composed of a broad-spectrum protease and a physiological buffer. The DNA extraction process can be efficiently completed by subjecting the sample and the lysate to a heat treatment for 10 minutes. The physiological buffer is preferably a Tris-EDTA buffer, and the pH value of the Tris-EDTA buffer is preferably 8.

[0080] In some embodiments, the Corynebacterium avium detection kit provided by the present invention comprises a lyophilization protective agent, and each component in the Corynebacterium avium detection composition is packaged in the form of a lyophilized powder, which can extend the shelf life of the kit, and the components can be mixed together when used. Alternatively, a system consisting of basic dry powder (recombinant enzyme, single-stranded binding protein, polymerase, ATP energy regeneration enzyme, signal amplification enzyme system), upstream primer, downstream primer, TaqMan fluorescent probe, activation solution NTP, buffer, RNase-free Water, and lyophilization protective agent is prepared into a pre-packed lyophilized tube.

[0081] When using this kit, follow the steps below:

[0082] S1. Collect samples, add lysis buffer, and incubate at 95°C for 10 minutes to obtain sample DNA;

[0083] S2. Add the sample DNA to the above kit for room temperature amplification reaction;

[0084] S3. The fluorescence signal Tt value is used as the detection result of Corynebacterium avium. If there is no Tt value in the cycle, it means that the sample to be tested is not infected with Corynebacterium avium, and the test result is negative. If there is a Tt value, it means that the sample to be tested is infected with Corynebacterium avium, and the test result is positive.

[0085] Optionally, step S2 is performed in a portable metal bath, and step S3 is detected using a portable fluorescence detector, and the detection is completed in 10 to 30 minutes. The kit of the present invention is used in conjunction with a portable metal bath and a portable fluorescence detector, and can be carried to any location such as a rural site for detection, meeting application scenarios such as environmental detection in experimental barriers and detection next to animal cages.

[0086] The Corynebacterium spp. detection device provided by the present invention comprises:

[0087] A DNA extraction module, which is used to extract sample DNA;

[0088] An amplification module, which is used to amplify the sample DNA using the Corynebacterium muridarum detection composition to obtain an amplification product;

[0089] The fluorescence detection module is used to detect the fluorescence signal of the amplification product.

[0090] The detection device is designed to provide users with a complete solution from sample processing to result analysis, making the detection process of Corynebacterium rapae more automated and standardized, greatly improving laboratory work efficiency and the reliability of test results.

[0091] In some embodiments, the Corynebacterium muridarum detection device provided by the present invention further includes a judgment module:

[0092] The judgment module judges whether the sample contains Corynebacterium ratum according to the detection result of the fluorescence detection module; if there is no fluorescence signal, it is judged that the sample does not contain Corynebacterium ratum; if there is a fluorescence signal, it is judged that the sample contains Corynebacterium ratum.

[0093] The technical solution of the present invention is described in detail below through specific embodiments:

[0094] Example 1

[0095] The Corynebacterium muridarum positive samples used in the test were from Shanghai Chaorui Biotechnology Co., Ltd., with the trade name: Corynebacterium muridarum fluorescent quantitative positive nucleic acid, PAER (DNA). The concentration was detected using a micro-spectrophotometer (Nanodrop) and then stored in a -80°C refrigerator.

[0096] Reaction system: in a mixture containing basic dry powder (200 μL of 20-200 ng / μL helicase, 60 μL of 1-30 ng / μL recombinase, 240 μL of 100-1000 ng / μL single-stranded binding protein, 96 μL of 10-100 ng / μL polymerase, 1-10 ng / μL 80 μL of ATP energy regeneration enzyme, signal amplifier enzyme group including: 200 μL of 1-30 ng / μL mouse coryneformis fluorescent quantitative positive nucleic acid restriction endonuclease, 60 μL of 1-30 ng / μL auxiliary protein), 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of TaqMan fluorescent probe (1 μM), 7 μL of mouse coryneformis fluorescent quantitative positive nucleic acid were added to the eight-tube, centrifuged instantly, and allowed to stand at room temperature for 2 minutes. 10 μL of activation solution (a solution composed of NTP (nucleoside triphosphate) and Tris-EDTA buffer) was added along the wall of the eight-tube, centrifuged instantly; oscillated and mixed for 10 seconds, centrifuged instantly, and then tested on the machine. The portable PCR instrument was used, the reaction temperature was 25°C, and the signal was collected once in each cycle, for a total of 30 cycles, the reaction time was 30 minutes, and the reporter group was set to FAM.

[0097] TaqMan fluorescent probe screening was performed using the fluorescent quantitative positive nucleic acid of Corynebacterium muris as a template, Tris-EDTA buffer (TE) as a negative control, and RNase-free Water (Takara, 9012) as a blank control. Primers F1, F4, and F6 were cross-combined with primers R1, R4, and R6, and TaqMan fluorescent probes RNA1, RNA2, RNA3, RNA4, RNA5, and RNA6 were added to the primers of F1R1, F4R1, F6R1, F1R4, F4R4, F6R4, F1R6, F4R6, and F6R6, respectively. The corresponding identical primer sets were compared, and the TaqMan fluorescent probe with the smallest Tt value and the highest endpoint fluorescence value was selected. At the same time, according to the TaqMan fluorescent probe, the screening results of the corresponding 4 groups of DNA primers were determined, and the TaqMan fluorescent probe with the smallest Tt value and the highest endpoint fluorescence value and the primer set with no amplification signal in the negative control were selected. The results showed that there were 54 combinations of the above 9 primers F1R1, F4R1, F6R1, F1R4, F4R4, F6R4, F1R6, F4R6, F6R6 and 6 TaqMan fluorescent probes RNA1, RNA2, RNA3, RNA4, RNA5 and RNA6, 18 of which had good amplification effects. Among them, the Tt value of the F6R4RNA6 combination was the lowest at 12.5, and the fluorescence value was relatively high. There was no amplification signal in the negative control, so RNA6 primers were selected, and the F6 RNA6 combination was used for subsequent experiments ( Figure 2-5 ).

[0098] Example 2

[0099] Using the fluorescent quantitative positive nucleic acid of Corynebacterium muris as a template, according to the reaction system of Example 1, the RNA6 primer and the upstream primer F6 were fixed, and F6 was screened with the downstream primers R1, R2, R3, R4, R5, and R6, respectively, to select the primer set with the smallest Tt value, the highest endpoint fluorescence value, and the negative control without amplification signal. The results showed that in the downstream primer screening results, the Tt value of the F6R1 RNA6 group was relatively low at 10.8, and the fluorescence value was relatively high, indicating that the downstream primer R1 had a better effect, the Tt value of the F6R5RNA6 group was 11.9, and R5 was the second choice, and there was no amplification signal in the negative control, so it was determined to select R1 and R5 primers, and the R1 RNA6 and R5RNA6 combinations were used to carry out subsequent experiments ( Figure 6 ).

[0100] Example 3

[0101] Using the fluorescent quantitative positive nucleic acid of Corynebacterium muris as a template, according to the reaction system of Example 1, RNA6 primer and downstream primers R1, R5 were fixed, and upstream primers F1, F2, F3, F4, F5, and F6 were selected for screening, and the DNA primers with the smallest Tt value and the highest endpoint fluorescence value and the primer set with no amplification signal in the negative control were selected. The results showed that in the upstream primer screening results, the Tt value of the F5R1RNA6 group was relatively low at 5.5, and the fluorescence value was relatively high. The Tt value of the F6R5 RNA6 group was 11.9, and the fluorescence value was the second highest, indicating that the upstream primer F5 had a better effect, and there was no amplification signal in the negative control, so it was determined to select F5 and F6 primers, and to determine to use the combination of F5R1RNA6 and F6R5RNA6 to carry out the experiment ( Figure 7 ).

[0102] Example 4

[0103] Using Gram-negative bacteria (G - ): Commercialized Corynebacterium muris fluorescent quantitative positive nucleic acid, PAER (DNA), Commercialized Salmonella fluorescent quantitative positive nucleic acid, PAER (DNA), Gram-positive bacteria (G + ): commercial Klebsiella pneumoniae fluorescence quantitative positive nucleic acid, PAER (DNA), commercial Bacillus pilosus fluorescence quantitative positive nucleic acid, PAER (DNA) as template, according to the reaction system of Example 1, TE as negative control, RNase-free Water as blank control, and the optimal primer group F5R1 RNA6 obtained by screening were specifically detected according to the reaction system, time and temperature of Example 1. The results showed that two Gram-negative bacteria (G-) and two Gram-positive bacteria (G+) were selected for specificity experiments. The three tested bacteria were not detected, and Corynebacterium muris was detected, indicating that the primer and probe combination screened by the present invention has high specificity ( Figure 7 )

[0104] Example 5

[0105] Reaction system: A system containing basic dry powder (recombinant enzyme, single-stranded binding protein, polymerase, ATP energy regeneration enzyme, signal amplification enzyme system), DNA upstream primers (F5, F6), DNA downstream primers (R1, R5), TaqMan fluorescent probe (RNA6), activation solution NTP (nucleoside triphosphate), buffer, RNase-free water, and freeze-drying protective agent is prepared into eight-unit freeze-dried tubes.

[0106] Experimental operation: Add 1 μL of Corynebacterium avium fluorescent quantitative positive nucleic acid upstream primer (10 μM), 1 μL of Corynebacterium avium fluorescent quantitative positive nucleic acid downstream primer (10 μM), 1 μL of TaqMan fluorescent probe (1 μM), and 7 μL of commercial positive nucleic acid into the eight-tube concatenation and centrifuge instantly. Let stand at room temperature for 2 min, add 10 μL of activation solution (composed of NTP (nucleoside triphosphate) and buffer) along the wall of the eight-tube concatenation and centrifuge instantly; oscillate to mix for 10 s, centrifuge instantly, and then detect on the machine.

[0107] Portable PCR instrument, reaction temperature 25 ° C, signal collection once per cycle, a total of 30 cycles, reaction time of 30 minutes, the reporter group is set to FAM.

[0108] The number of commercial positive nucleic acid copies is 1×10 6 , the nucleic acid was diluted with RNase-free water to select a copy number of 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 10, 5, 1 were tested on the machine according to the reaction system, and the optimal primer groups F5R1 RNA6 and F6R5 RNA6 were screened for sensitivity testing. The results showed that the primer probe screened by F5R1 RNA6 could detect a minimum copy number of 1, with high sensitivity, and the gradient diluted nucleic acid had a good effect on the machine ( Figure 8-9 ).

[0109] Example 6

[0110] Commercial Salmonella positive nucleic acid (hereinafter referred to as P1), commercial Klebsiella pneumoniae positive nucleic acid (hereinafter referred to as P2), Corynebacterium muris DNA as positive control, TE as negative control, RNase-free water as blank control, and the optimal primer set F5R1 RNA6 obtained by screening were used to judge the effect of this method in the sample according to the reaction system, time and temperature of Example 5. In addition, the sample was used for qPCR detection. The results showed that it was consistent with the detection results of qPCR, with an accuracy rate of 100% (Table 1 and Fig. 9 ).

[0111] Example 7

[0112] The feces of five SPF-grade C57 mice were used as test samples, and the sample DNA was quickly extracted using a lysis buffer to obtain samples A, B, C, D, and E. The DNA of Corynebacterium muridarum was used as a positive control, and TE was used as a negative control to determine the effectiveness of this method in on-site detection of actual samples. According to the reaction system of Example 5, a portable fluorescence detector was used for detection. After 5 samples were drawn in the laboratory, they were tested on-site, and the sample DNA was quickly extracted using a lysis buffer. The remaining samples were used for qPCR detection. The results showed that the effect of rapid detection on site was consistent with the detection results of qPCR, with an accuracy rate of 100% (Table 2 and Fig.10 ).

[0113] Table 1. Comparison of the results of the present invention and qPCR detection method

[0114]

[0115] Table 2. Comparison of the results of the present invention and qPCR detection method

[0116]

[0117] Table 3 is a primer screening table of the present invention

[0118]

[0119]

[0120] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A primer set for RAA amplification of Corynebacterium muris nucleic acid, characterized in that: including an upstream primer and a downstream primer of a target double-stranded DNA; The nucleotide sequence of the target double-stranded DNA is shown in SEQ ID NO.19; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.:

7.

2. A composition for detecting Corynebacterium muris, characterized in that: The Corynebacterium muridarum detection composition comprises: An upstream primer and a downstream primer of the target double-stranded DNA, wherein the upstream primer comprises a T7 promoter; A recombinase that can catalyze the formation of a double-stranded DNA containing a T7 promoter by an upstream primer and a first single-stranded target double-stranded DNA; T7 RNA polymerase, which is used to catalyze the transcription of double-stranded DNA containing a T7 promoter to generate single-stranded RNA; Transcriptase, which catalyzes the reverse transcription of single-stranded RNA into double-stranded DNA / RNA; ATP energy regenerating enzyme, which is used to provide ATP energy in DNA recombination and transcription processes; A helicase, which is used to catalyze the hydrolysis of the DNA / RNA double strand into RNA single strand and DNA single strand; Single-strand binding protein, which is used to bind to single-stranded DNA and prevent the self-polymerization of single-stranded DNA; TaqMan fluorescent probe, which includes an RNA chain that can bind to a single-stranded DNA, and the 5' end and 3' end of the RNA chain have a reporter group and a quencher group respectively; A cleavage enzyme, which is used to cleave the RNA chain of the TaqMan fluorescent probe to release the fluorescent group when the TaqMan fluorescent probe is bound to the DNA single chain; in: The nucleotide sequence of the target double-stranded DNA is shown in SEQ ID NO.19; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.: 7; The nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.:

18.

3. The composition for detecting Corynebacterium avium according to claim 2, characterized in that: The quenching group of the TaqMan fluorescent probe is one of BHQ, BHQ1, BHQ2, and TAMRA, and the reporting group is one of FAM, FAM-X, CY3, CY5, HEX, and TET.

4. The composition for detecting Corynebacterium muris according to claim 2, characterized in that: The Corynebacterium muridarum detection composition also includes a plasmid standard of the target double-stranded DNA.

5. The composition for detecting Corynebacterium avium according to claim 2, characterized in that: The Corynebacterium murinus detection composition is used for amplifying and detecting target double-stranded DNA under a constant temperature condition of 25°C.

6. A kit for detecting Corynebacterium muris, characterized in that: The Corynebacterium muridarum detection kit comprises the Corynebacterium muridarum detection composition according to any one of claims 2 to 5.

7. The kit for detecting Corynebacterium avium according to claim 6, characterized in that: The Corynebacterium muridarum detection kit further comprises a lysis solution, which comprises a broad-spectrum protease and a physiological buffer.

8. The Corynebacterium spp. detection kit according to claim 6 or 7, characterized in that: The Corynebacterium muridarum detection kit comprises a freeze-dried protective agent, and each component in the Corynebacterium muridarum detection composition is packaged in the form of freeze-dried powder.

9. A Corynebacterium muridarum detection device, characterized in that: include: A DNA extraction module, which is used to extract sample DNA; An amplification module, which is used to amplify sample DNA using the Corynebacterium muridarum detection composition according to any one of claims 2 to 5 to obtain an amplification product; The fluorescence detection module is used to detect the fluorescence signal of the amplification product.

10. The device for detecting Corynebacterium muridarum according to claim 9, characterized in that: The Corynebacterium muridarum detection device further comprises a judgment module: The judging module judges whether the sample contains Corynebacterium ratum according to the detection result of the fluorescence detection module; if there is no fluorescence signal, it is judged that the sample does not contain Corynebacterium ratum; if there is a fluorescence signal, it is judged that the sample contains Corynebacterium ratum.

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