CN110923346A

CN110923346A - Primer, probe, kit and detection method for RNA isothermal amplification detection of mycoplasma bovis

Info

Publication number: CN110923346A
Application number: CN201911315618.6A
Authority: CN
Inventors: 刘喜富; 乌日琴; 张銮
Original assignee: Zhangjiakou Jian Yuan Technology Co Ltd
Current assignee: Zhangjiakou Jian Yuan Technology Co Ltd
Priority date: 2019-09-02
Filing date: 2019-12-19
Publication date: 2020-03-27

Abstract

The invention discloses a primer, a probe, a kit and a detection method for RNA isothermal amplification detection of mycoplasma bovis. The specific detection primer and the probe, the detection kit containing the detection primer and the probe, and the detection method using the detection kit to carry out RNA isothermal amplification have the characteristics of high sensitivity, strong specificity, low pollution (RNA of an amplification product is easy to degrade in a natural environment), rapidness, low requirement on instruments, simple operation process, suitability for self-inspection of food enterprises and basic detection mechanisms, suitability for field detection, and particularly suitability for rapid detection of cow milk and guarantee of cow safety.

Description

Primer, probe, kit and detection method for RNA isothermal amplification detection of mycoplasma bovis

Technical Field

The invention relates to the technical field of pathogenic bacteria microorganism detection, in particular to a rapid detection technology for detecting mycoplasma bovis based on RNA isothermal amplification, and particularly relates to a specific detection primer, a probe, a kit and a detection method for detecting mycoplasma bovis.

Background

Mycoplasma Bovis (MB), belonging to the genus Mycoplasma, has recently led to the rapid development of the large-scale cattle industry due to the vigorous development of milk, and has attracted extensive attention of people, such as Mycoplasma bovis, which poses a great threat to the cattle industry, can cause cattle to produce diseases such as pneumonia, mastitis, arthritis, genital tract inflammation, and the like, and can seriously cause cattle abortion and infertility, especially the caused bovine pneumonic plague is a virulent contagious disease. Therefore, the rapid detection of the mycoplasma bovis in the cow milk not only can effectively prevent and treat related sick cows in time and reduce loss, but also is a necessary means for ensuring the vigorous development of the cow breeding industry.

In recent years, the rapid detection and identification methods for food-borne pathogenic bacteria have been developed rapidly, and the rapid detection methods include an immunology-based ELISA technology, an immunomagnetic bead technology, a full-automatic immunoenzyme labeling detection series, an immunocolloidal gold technology and the like, a molecular biology-based RNA fingerprint technology, a real-time fluorescence PCR technology, a gene chip technology and the like, and a paper method-based enzyme-contact reaction technology for bacterial metabolism and the like. The detection of the mycoplasma bovis by the national standard culture method needs 4-7 days, the detection of the mycoplasma bovis by the standard real-time fluorescence PCR method needs 2-5 days, the detection of the mycoplasma bovis by the real-time fluorescence isothermal Amplification (SAT) needs 6 hours, and the Amplification technology Based on Nucleic Acid Sequences (NASBA) and the Transcription-Mediated Amplification Technology (TMA) have no related mycoplasma bovis RNA detection kit temporarily.

The traditional mycoplasma bovis detection method has the disadvantages of low bacteria concentration, high culture condition requirement, long consumed time and low detection efficiency, so that mycoplasma bovis cannot be monitored in time, and field detection cannot be realized.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a primer, a probe, a kit and a detection method for RNA isothermal amplification detection of mycoplasma bovis, so that the rapid and accurate detection of the mycoplasma bovis on site is realized. According to the method of the invention, the detection takes only about 150 min.

In order to achieve the purpose, the invention adopts the following technical scheme:

a primer and a probe for RNA isothermal amplification detection of mycoplasma bovis, wherein the primer comprises: the upstream primer MBnT7 comprises: CTGACCTTCGGGTAAAGC, or TGGTTTGTCGCGTTGTTC, or a homologous sequence thereof; the downstream primer MB T7 comprises: a T7 promoter sequence and a target primer sequence; wherein the target primer sequence comprises at least 8 nucleotide sequences of CCACACCGCT AGAGC, or ACCAGGAATTCCAGT, or homologous sequences thereof; wherein the T7 promoter sequence comprises at least 10 nucleotide sequences of AATTTAATAC AGCTCACTAT AGGGAGA, or a homologous sequence thereof; wherein the probe has a hairpin structure with a loop and a stem that is self-complementary.

According to some embodiments of the invention, the probe is covalently labeled at both ends with a fluorophore and a quencher, respectively, preferably the fluorophore comprises FAM, HEX or VIC and the quencher comprises DABCYL.

According to certain embodiments of the invention, the probe sequence comprises at least 10 nucleotides of CUGCGGGACCCAGGGAUGCAUGUCGCAG, or CTGCG AGCGTG CGGGCGATAC GGG CAG CGCAG, or a homologous sequence thereof.

According to some embodiments of the invention, the homologous sequence refers to a nucleotide sequence that is at least 70% identical to the sequence.

According to certain embodiments of the invention, the homologous sequences have substantially the same activity as the sequences disclosed herein.

A kit for detecting mycoplasma bovis by RNA isothermal amplification comprises a reaction solution, a detection solution containing the primers and the probes, and an enzyme solution containing T7RNA polymerase and M-MLV reverse transcriptase.

According to some embodiments of the present invention, the reaction solution comprises 2-10 mmol/L Tris HCl, 20-40 mmol/L MgCl₂，0.5～5mM dNTPs，1～10mM NTPs，2.5～2.7mmol/L KCl，2～10mmol/LNa₂HPO₄，0.5～2mmol/L KH₂PO₄。

According to some embodiments of the invention, the detection solution comprises 5-15 pmol/L upstream primer MB nT7, 5-15 pmol/L downstream primer MB T7, 5-10 pmol/L MB fluorescent probe, 1-5 mmol/L Tris-HCl pH8.0, 1-5 mmol/L EDTA, and the balance DEPC water.

According to some embodiments of the invention, the enzyme solution comprises 500 to 1000U/reaction M-MLV reverse transcriptase, 500 to 1500U/reaction T7RNA polymerase, 0.5 to 1mmol/L Tris-HCl pH8.0, 2.5 to 2.7mmol/L KCl, 0.5 to 1mmol/L EDTA, 2 to 10% BSA.

According to some embodiments of the invention, the kit comprises:

reaction solution: the reaction solution contains 2-10 mmol/L Tris HCl, 20-40 mmol/L MgCl₂，0.5～5mMdNTPs，1～10mM NTPs，2.5～2.7mmol/L KCl，2～10mmol/L Na₂HPO₄，0.5～2mmol/L KH₂PO₄(ii) a Detection liquid: the detection solution comprises 5-15 pmol/L of an upstream primer MB nT7, 5-15 pmol/L of a downstream primer MB T7, 5-10 pmol/L of an MB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/L EDTA and the balance of DEPC water; and

enzyme solution: the enzyme solution comprises 500-1000U/reaction M-MLV reverse transcriptase, 500-1500U/reaction T7RNA polymerase, 0.5-1 mmol/L Tris-HCl pH8.0, 2.5-2.7 mmol/L KCl, 0.5-1 mmol/L EDTA and 2-10% BSA.

According to some embodiments of the invention, the kit comprises:

reaction solution: the reaction solution contains 10mmol/L Tris-HCl and 40mmol/L MgCl₂，2.5mM dNTPs，6mMNTPs，2.7mmol/L KCl，10mmol/L Na₂HPO₄，2mmol/L KH₂PO₄；

Detection liquid: the detection solution contains 10pmol/L of upstream primer MB nT7, 10pmol/L of downstream primer MB T7, 8pmol/L of fluorescent Probe MB Probe, 1mmol/L of LTris-HCl pH8.0, 0.25mM EDTA and DEPC water.

Enzyme solution: the enzyme solution comprises 1000U/reaction M-MLV reverse transcriptase, 1500U/reaction T7RNA polymerase, 1mmol/L Tris-HCl pH8.0, 2.7mmol/L KCl, 1mmol/L EDTA, and 10% BSA.

According to some embodiments of the invention, the kit further comprises a lysis solution, a nucleic acid extraction solution, a washing solution, a positive control and a negative control.

According to some embodiments of the invention, the lysate comprises 10% to 20% Triton X-100, 2mol/L to 10mol/L guanidinium isothiocyanate, 2 to 10mmol/L Tris HCl, pH2.0 to pH 9.0.

According to some embodiments of the present invention, the nucleic acid extract comprises 40-200 mM EDTA, 50-500 mg/L magnetic beads, and 1-50. mu.M capture probes.

According to some embodiments of the invention, the extraction solution comprises a capture probe that is oligo (dT).

According to some embodiments of the invention, the oligo (dT) is 10-26 in length.

According to some embodiments of the invention, the oligo (dT) is 18 in length.

According to some embodiments of the invention, the washing solution comprises 2-2.5 mmol/L NaCl, 2-10 mol/L guanidinium isothiocyanate, 2-10 mmol/L Tris HCl, pH 7.0-9.0, 2-10% SDS.

According to certain embodiments of the invention, the positive control comprises 10⁴～10⁶copies/mL Mycoplasma bovis RNA.

According to some embodiments of the invention, the negative control comprises saline.

According to some embodiments of the invention, the kit further comprises:

lysis solution: the lysis solution comprises 10-20% of Triton X-100, 2-10 mol/L of guanidinium isothiocyanate, 2-10 mmol/L of Tris HCl and pH 2.0-9.0;

nucleic acid extracting solution: the nucleic acid extracting solution comprises 40-200 mM EDTA, 50-500 mg/L magnetic beads and 1-50 mu M capture probes;

washing liquid: the washing solution comprises 2-2.5 mmol/L NaCl, 2-10 mol/L guanidinium isothiocyanate, 2-10 mmol/L Tris HCl, pH7.0-9.0, 2-10% SDS;

positive control: the positive control comprises 10⁴～10⁶copy/mL mycoplasma bovis RNA; and

negative control: the negative control comprises saline.

According to some embodiments of the invention, the kit further comprises:

nucleic acid extracting solution: the nucleic acid extracting solution comprises 40-200 mM EDTA, 50-500 mg/L magnetic beads and 1-50 mu M capture probes, and preferably the capture probes are oligo (dT);

negative control: the negative control comprises saline.

According to some embodiments of the invention, the kit further comprises:

nucleic acid extracting solution: the nucleic acid extracting solution comprises 40-200 mM EDTA, 50-500 mg/L magnetic beads and 1-50 mu M capture probes, preferably the capture probes are oligo (dT), and preferably the length of oligo (dT) is 10-26;

washing liquid: the washing solution comprises 2-2.5 mmol/L NaCl, 2-10 mol/L guanidinium isothiocyanate, 2-10 mmol/L Tris HCl, pH 7.0-9.0 and 2-10% SDS;

negative control: the negative control comprises saline.

According to some embodiments of the invention, the kit comprises:

reaction solution: the reaction solution contains 2-10 mmol/L Tris HCl, 20-40 mmol/L MgCl₂，0.5～5mMdNTPs，1～10mM NTPs，2.5～2.7mmol/L KCl，2～10mmol/L Na₂HPO₄，0.5～2mmol/L KH₂PO₄；

Detection liquid: the detection solution comprises 5-15 pmol/L of an upstream primer MB nT7, 5-15 pmol/L of a downstream primer MBT7, 5-10 pmol/L of an MB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/LEDTA and the balance of DEPC water;

enzyme solution: the enzyme solution comprises 500-1000U/reaction M-MLV reverse transcriptase, 500-1500U/reaction T7RNA polymerase, 0.5-1 mmol/L Tris-HCl pH8.0, 2.5-2.7 mmol/L KCl, 0.5-1 mmol/L EDTA and 2-10% BSA;

washing liquid: the washing solution comprises 2-2.5 mmol/L NaCl, 2-10 mol/L guanidinium isothiocyanate, 2-10 mmol/L Tris HCL, pH7.0-9.0, 2% -10% SDS;

positive control: the positive control comprises 10⁴～10⁶copies/mL Mycoplasma bovis RNA(ii) a And

negative control: the negative control comprises saline.

According to some embodiments of the invention, the kit comprises:

Detection liquid: the detection solution comprises 5-15 pmol/L of an upstream primer MB nT7, 5-15 pmol/L of a downstream primer MBT7, 5-10 pmol/L of an MB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/L EDTA and the balance of DEPC water;

negative control: the negative control comprises saline.

According to some embodiments of the invention, the kit comprises:

reaction solution: the reaction solution contains 2-10 mmol/L TrisHCl and 20-40 mmol/L MgCl₂，0.5～5mMdNTPs，1～10mM NTPs，2.5～2.7mmol/L KCl，2～10mmol/L Na₂HPO₄，0.5～2mmol/LKH₂PO₄；

negative control: the negative control comprises saline.

According to some embodiments of the invention, the kit comprises:

reaction solution: 10mmol/L Tris-HCl, 40mmol/L MgCl₂，2.5mM dNTPs，6mMNTPs，2.7mmol/LKCl，10mmol/L Na₂HPO₄，2mmol/L KH₂PO₄；

Detection liquid: 10pmol/L of upstream primer MB nT7, 10pmol/L of downstream primer MB T7, 8pmol/L of fluorescent probe MBProbe, 1mmol/L of Tris-HCl pH8.0, 0.25mM EDTA, DEPC water.

Enzyme solution: 1000U/reaction M-MLV reverse transcriptase, 1500U/reaction T7RNA polymerase, 1mmol/L Tris-HClpH8.0, 2.7mmol/L KCl, 1mmol/L EDTA, 10% BSA;

lysis solution: 10% TritonX-100, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH 8.0;

nucleic acid extracting solution: 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe;

washing liquid: 2mmol/L NaCl, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH8.0, 10% SDS;

positive control: comprises 10⁵copy/mL mycoplasma bovis RNA;

negative control: physiological saline.

According to some embodiments of the invention, the kit comprises:

reaction solution: 10mmol/L Tris-HCl, 40mmol/L MgCl₂，2.5mM dNTPs，6mM NTPs，2.7mmol/L KCl，10mmol/L Na₂HPO₄，2mmol/L KH₂PO₄；

nucleic acid extracting solution: 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe; the length of oligo (dT) is 18;

positive control: comprises 10⁵copy/mL mycoplasma bovis RNA;

negative control: physiological saline.

The invention also provides a method for detecting mycoplasma bovis by using the kit, which comprises the following steps:

(1) extracting target RNA in a sample to be detected;

(2) performing RNA isothermal amplification on target RNA in a sample to be detected by using the primer and the probe;

(3) recording the ct value of the reaction of the sample to be detected: the sample with ct less than or equal to 55 is positive; samples 55 < ct < 60 are recommended for retesting, and the retest results are: samples with ct < 60 are positive; samples with ct no value or 60 were negative.

According to some embodiments of the invention, the detection uses a FAM channel, a HEX channel, or a VIC channel.

According to some embodiments of the invention, the reaction system for amplification is a mixture of a reaction solution and a detection solution.

According to some embodiments of the invention, the amplification reaction condition is 42 ℃ +/-1 ℃ in a fluorescence PCR instrument for 40-60 min.

According to some embodiments of the present invention, the extracting the target RNA from the sample to be detected comprises (1) lysing the thallus in the sample to be detected with the lysis solution to obtain a lysed thallus containing the target RNA; and (2) adding the nucleic acid extracting solution into the lysis solution obtained in the step (1), so that the capture probes on the magnetic beads are combined with the target RNA, washing with the washing solution, and removing the nucleic acids which are not combined with the magnetic beads to obtain the target RNA.

According to some embodiments of the present invention, the isothermal amplification of the target RNA in the sample to be detected by using the primers and the probe comprises adding the target RNA extracted in step (2) to an amplification reaction solution comprising the reaction solution and the detection solution, performing a first incubation and a second incubation, wherein the first incubation is performed at 62 ℃ ± 2 ℃ for 5-10 min, the second incubation is performed at 40 ± 1 ℃ for 5-10 min, then adding the enzyme solution, reacting at 42 ℃ ± 1 ℃ for 40-60 min, and synchronously recording the change of the fluorescence signal by using the detector.

According to some embodiments of the invention, the method comprises:

(1) cracking thalli in a sample to be detected by using the lysate to obtain a cracking solution containing target RNA; adding the nucleic acid extracting solution into a lysis solution to combine the capture probes on the magnetic beads with the target RNA, washing with the washing solution, and removing nucleic acids which are not combined with the magnetic beads to obtain the target RNA;

(2) adding the target RNA extracted in the step (1) into an amplification reaction solution consisting of the reaction solution and the detection solution, performing first incubation and second incubation, wherein the first incubation is performed at 62 +/-2 ℃ for 5-10 min, the second incubation is performed at 40 +/-1 ℃ for 5-10 min, then adding the enzyme solution, reacting at 42 +/-1 ℃ for 40-60 min, and synchronously recording the change of a fluorescence signal by using a detector;

(3) and recording the ct value of the reaction of the sample to be detected according to the time and the intensity of the generated fluorescent signal: the sample with ct less than or equal to 55 is positive; samples 55 < ct < 60 are recommended for retesting, and the retest results are: samples with ct < 60 are positive; samples with ct no value or 60 were negative.

According to some embodiments of the invention, the test sample is tested and determined with reference to the positive control and negative control results; the judging method comprises the following steps: and if the detection result is positive, the sample contains mycoplasma bovis, and if the detection result is negative, the sample does not contain mycoplasma bovis.

Detailed Description

Primer and probe for detecting mycoplasma bovis by RNA isothermal amplification

The invention provides a primer and a probe for RNA isothermal amplification detection of mycoplasma bovis, wherein a downstream primer of the primer comprises a T7 promoter.

According to some embodiments of the invention, the upstream primer MB nT7 (primer that does not comprise a promoter) sequence comprises: CTGACCTTCGGGTAAAGC, or TGGTTTGTCGCGTTGTTC, or a homologous sequence thereof; the sequence of the downstream primer MB T7 (primer comprising T7 promoter) comprises: a T7 promoter sequence, and a target primer sequence; wherein the target primer sequence comprises at least 8 nucleotide sequences of CCACACCGCT AGAGC, or ACCAGGAATTCCAGT, or homologous sequences thereof; wherein the T7 promoter sequence comprises at least 10 nucleotide sequences of AATTTAATAC AGCTCACTAT AGGGAGA, or a homologous sequence thereof.

According to certain embodiments of the invention, the homologous sequences have a nucleotide sequence that is at least 70% identical, such as a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% identical.

According to certain embodiments of the invention, the forward primer MBnT7 sequence comprises a nucleotide sequence of at least 10, such as at least 12, at least 14, at least 15, at least 16, at least 17, at least 18.

According to certain embodiments of the invention, the target primer sequence in the downstream primer MB T7 comprises a nucleotide sequence of at least 8, such as at least 8, at least 10, at least 12, at least 13, at least 14, at least 15.

According to certain embodiments of the invention, the T7 promoter sequence in the downstream primer MB T7 comprises at least 10 nucleotide sequences, such as at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 25, at least 26, at least 27 nucleotide sequences.

According to some embodiments of the invention, in the downstream primer MB T7, the T7 promoter sequence is located at the 5' end of the target primer sequence.

According to some embodiments of the invention, in the downstream primer MB T7, the T7 promoter sequence is located at the 3' end of the target primer sequence.

According to some embodiments of the invention, the primer sequence is:

the upstream primer is as follows: CTGACCTTCGGGTAAAGC, respectively;

the downstream primer is: AATTTAATAC AGCTCACTAT AGGGAGA CCACACCGCT AGAGC are provided.

According to some embodiments of the invention, the probe is a fluorescent probe having the sequence: CUGCGGGACCCAGGGAUGCAUGUCGCAG are provided.

According to some embodiments of the invention, the probe is a fluorescent probe.

According to some embodiments of the invention, the probe is a molecular beacon.

According to some embodiments of the invention, the probe has a hairpin structure with a loop and a stem that is self-complementary.

According to some embodiments of the invention, the probe has a hairpin structure of one loop and one self-complementary stem.

According to certain embodiments of the invention, the loop portion of the probe is complementary to the target.

According to some embodiments of the invention, the probe is labeled with a fluorophore and a quencher at both ends thereof.

According to some embodiments of the invention, the probe is covalently labeled with a fluorophore and a quencher at both ends, respectively.

According to some embodiments of the invention, the fluorescent probe is a molecular beacon having a hairpin structure of a loop and a self-complementary stem, and consisting of a single-stranded nucleic acid molecule covalently labeled with a fluorescent dye and a quencher at each end, wherein the loop portion of the molecular beacon is complementary to the target and the two ends are stem due to the complementarity.

According to some embodiments of the invention, the probe has a hairpin structure of a loop and a self-complementary stem, consisting of a single-stranded nucleic acid molecule covalently labeled at both ends with a fluorescent dye and a quencher, respectively, the loop portion of the molecular beacon being complementary to the target, and the two ends being stem due to the complementarity.

According to some embodiments of the invention, the probe sequence comprises: CUGCGGGACCCAGGGAUGCAUGUCGCAG, or CTGCG AGCGTG CGGGCGATAC GGG CAG CGCAG, or a homologous sequence thereof.

According to certain embodiments of the invention, the probe sequence comprises a nucleotide sequence of at least 10, such as at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 27, at least 30, at least 32.

Preferably, the 5 'end of the probe contains a fluorescent group, and the 3' end of the probe contains a quenching group.

Preferably, the 5 'end of the probe contains a quenching group, and the 3' end of the probe contains a fluorescent group.

Preferably, the fluorophore comprises FAM, HEX or VIC.

Preferably, the quencher group comprises DABCYL.

Preferably, the 5 'end of the probe is labeled with FAM, HEX or VIC, and the 3' end is labeled with DABCYL. Preferably, the fluorescent probe is labeled with DABCYL at the 5 'end and FAM, HEX or VIC at the 3' end.

Reagent kit

The invention also provides a kit for detecting mycoplasma bovis by RNA isothermal amplification, which comprises reaction liquid, detection liquid containing the primers and the probes in the technical scheme, and enzyme liquid containing T7RNA polymerase and M-MLV reverse transcriptase.

In the present invention, a solution composed of the reaction solution and the detection solution is also referred to as an amplification reaction solution.

Preferably, the kit further comprises: lysis solution, nucleic acid extracting solution, washing solution, positive control and negative control.

Reaction solution

Preferably, the reaction solution contains 2-10 mmol/L Tris HCl and 20-40 mmol/L MgCl₂，0.5～5mMdNTPs，1～10mM NTPs，2.5～2.7mmol/L KCl，2～10mmol/L Na₂HPO₄，0.5～2mmol/L KH₂PO₄。

According to some embodiments of the invention, the reaction solution comprises 2 to 10mmol/L Tris HCl, such as 4 to 10mmol/L, 6 to 10mmol/L, 8 to 10mmol/L, 2 to 8mmol/L, 4 to 8mmol/L, 6 to 8mmol/L, 2 to 6mmol/L, 4 to 6mmol/L or 2 to 4mmol/L LTris HCl. According to certain embodiments of the invention, the reaction solution comprises 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10mmol/L Tris HCl.

According to some embodiments of the invention, the reaction solution comprises 20 to 40mmol/L MgCl₂For example, 20 to 35mmol/L, 20 to 30mmol/L, 20 to 25mmol/L, 25 to 40mmol/L, 25 to 35mmol/L, 25 to 30mmol/L, 30 to 40mmol/L, 30 to 35mmol/L or 35 to 40mmol/LMgCl₂. According to certain embodiments of the invention, the reaction solution comprises 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/LMgCl₂。

According to some embodiments of the present invention, the reaction solution contains 0.5 to 5mM dNTPs, such as 0.5 to 4mM, 0.5 to 3mM, 0.5 to 2mM, 0.5 to 1mM, 1 to 5mM, 1 to 4mM, 1 to 3mM, 1 to 2mM, 2 to 5mM, 2 to 4mM, 2 to 3mM, 3 to 5mM, 3 to 4mM, or 4 to 5mM dNTPs. According to certain embodiments of the invention, the reaction solution comprises 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM or 5mM dNTPs.

According to some embodiments of the present invention, the reaction solution contains 1-10 mM NTPs, such as 1-8 mM, 1-5 mM, 1-3 mM, 3-10 mM, 3-8 mM, 3-5 mM, 5-10 mM, 5-8 mM, or 8-10 mM NTPs. According to certain embodiments of the invention, the reaction solution comprises 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM or 10mM NTPs.

According to certain embodiments of the invention, the reaction solution comprises 2.5 to 2.7mmol/L KCl, such as 2.5 to 2.6mmol/L or 2.6 to 2.7 mmol/LKCl. According to certain embodiments of the invention, the reaction solution comprises 2.5mmol/L, 2.51mmol/L, 2.52mmol/L, 2.53mmol/L, 2.54mmol/L, 2.55mmol/L, 2.56mmol/L, 2.57mmol/L, 2.58mmol/L, 2.59mmol/L, 2.6mmol/L, 2.61mmol/L, 2.62mmol/L, 2.63mmol/L, 2.64mmol/L, 2.65mmol/L, 2.66mmol/L, 2.67mmol/L, 2.68mmol/L, 2.69mmol/L or 2.7 mmol/LKCl.

According to some embodiments of the invention, the reaction solution comprises 2 to 10mmol/LNa₂HPO₄For example, 4 to 10mmol/L, 6 to 10mmol/L, 8 to 10mmol/L, 2 to 8mmol/L, 4 to 8mmol/L, 6 to 8mmol/L, 2 to 6mmol/L, 4 to 6mmol/L or 2 to 4mmol/LNa₂HPO₄. According to certain embodiments of the invention, the reaction solution comprises 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10mmol/L Na₂HPO₄。

According to some embodiments of the invention, the reaction solution comprises 0.5 to 2mmol/LKH₂PO₄For example, 0.5 to 1.5mmol/L, 0.5 to 1.0mmol/L, 1.0 to 2mmol/L, 1.0 to 1.5mmol/L, 1.5 to 2mmol/LKH₂PO₄. According to certain embodiments of the invention, the reaction solution comprises 0.5mmol/L, 0.6mmol/L, 0.7mmol/L, 0.8mmol/L, 0.9mmol/L, 1mmol/L, 1.1mmol/L, 1.2mmol/L, 1.3mmol/L, 1.4mmol/L, 1.5mmol/L, 1.6mmol/L, 1.7mmol/L, 1.8mmol/L, 1.9mmol/L, or 2mmol/LKH₂PO₄。

According to some embodiments of the invention, the reaction solution comprises: 10mmol/L Tris-HCl, 40mmol/LMgCl₂，2.5mM dNTPs，6mM NTPs，2.7mmol/L KCl，10mmol/L Na₂HPO₄，2mmol/L KH₂PO₄。

Detection solution containing primer and probe

Preferably, the detection solution comprises 5-15 pmol/L of upstream primer MB nT7, 5-15 pmol/L of downstream primer MBT7, 5-10 pmol/LMB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/L of EDTA, and the balance of DEPC water.

According to some embodiments of the invention, the detection solution comprises 5 to 15pmol/L of the upstream primer MB nT7, such as 5 to 12pmol/L, 5 to 10pmol/L, 5 to 8pmol/L, 8 to 15pmol/L, 8 to 12pmol/L, 8 to 10pmol/L, 10 to 15pmol/L, 10 to 12pmol/L, 12 to 15pmol/L of the upstream primer MB nT 7. According to some embodiments of the invention, the detection solution comprises 5pmol/L, 6pmol/L, 7pmol/L, 8pmol/L, 9pmol/L, 10pmol/L, 11pmol/L, 12pmol/L, 13pmol/L, 14pmol/L or 15pmol/L of the upstream primer MB nT 7.

According to some embodiments of the invention, the detection solution comprises 5 to 15pmol/L of the downstream primer MB T7, such as 5 to 12pmol/L, 5 to 10pmol/L, 5 to 8pmol/L, 8 to 15pmol/L, 8 to 12pmol/L, 8 to 10pmol/L, 10 to 15pmol/L, 10 to 12pmol/L, 12 to 15pmol/L of the downstream primer MB T7. According to some embodiments of the invention, the detection solution comprises 5pmol/L, 6pmol/L, 7pmol/L, 8pmol/L, 9pmol/L, 10pmol/L, 11pmol/L, 12pmol/L, 13pmol/L, 14pmol/L or 15pmol/L of the downstream primer MB T7.

According to some embodiments of the invention, the detection solution comprises 5 to 10pmol/LMB fluorescent probe, such as 5 to 8pmol/L or 8 to 10pmol/LMB fluorescent probe. According to some embodiments of the invention, the detection solution comprises 5pmol/L, 6pmol/L, 7pmol/L, 8pmol/L, 9pmol/L, or 10pmol/LMB fluorescent probes.

According to certain embodiments of the invention, the detection solution comprises 1-5 mmol/L Tris-HCl pH8.0, such as 1-2.5 mmol/L or 2.5-5 mmol/L LTris-HCl pH 8.0. According to certain embodiments of the invention, the detection solution comprises 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, 3mmol/L, 3.5mmol/L, 4mmol/L, 4.5mmol/L or 5mmol/L of LTris-HCl pH 8.0.

According to some embodiments of the invention, the detection solution comprises 1-5 mmol/L EDTA, such as 1-2.5 mmol/L or 2.5-5 mmol/L EDTA. According to certain embodiments of the invention, the detection solution comprises 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, 3mmol/L, 3.5mmol/L, 4mmol/L, 4.5mmol/L or 5 mmol/LEDTA.

According to some embodiments of the invention, the detection solution comprises 10pmol/L of upstream primer MB nT7, 10pmol/L of downstream primer MB T7, 8pmol/L of fluorescent Probe MB Probe, 1mmol/L of Tris-HCl pH8.0, 0.25mM EDTA, and the balance DEPC water.

Enzyme solution containing T7RNA polymerase and M-MLV reverse transcriptase

Preferably, the enzyme solution comprises 500-1000U/reaction M-MLV reverse transcriptase, 500-1500U/reaction T7RNA polymerase, 0.5-1 mmol/L Tris-HClpH8.0, 2.5-2.7 mmol/L KCl, 0.5-1 mmol/L EDTA and 2-10% BSA.

According to some embodiments of the present invention, the enzyme solution comprises 500-1000U/reaction M-MLV reverse transcriptase, such as 500-900U/reaction, 500-800U/reaction, 500-700U/reaction, 500-600U/reaction, 600-1000U/reaction, 600-900U/reaction, 600-800U/reaction, 600-700U/reaction, 700-1000U/reaction, 700-900U/reaction, 700-800U/reaction, 800-1000U/reaction, 800-900U/reaction, or 900-1000U/reaction M-MLV reverse transcriptase. According to some embodiments of the invention, the enzyme solution comprises 500U/reaction, 550U/reaction, 600U/reaction, 650U/reaction, 700U/reaction, 750U/reaction, 800U/reaction, 850U/reaction, 900U/reaction, 950U/reaction or 1000U/reaction M-MLV reverse transcriptase.

According to some embodiments of the present invention, the enzyme solution comprises 500-1500U/reaction T7RNA polymerase, such as 500-1200U/reaction, 500-1000U/reaction, 500-800U/reaction, 800-1500U/reaction, 800-1200U/reaction, 800-1000U/reaction, 1000-1500U/reaction, 1000-1200U/reaction, or 1200-1500U/reaction T7RNA polymerase. According to some embodiments of the invention, the enzyme solution comprises 500U/reaction, 600U/reaction, 700U/reaction, 800U/reaction, 900U/reaction, 1000U/reaction, 1100U/reaction, 1200U/reaction, 1300U/reaction, 1400U/reaction or 1500U/reaction T7RNA polymerase.

According to certain embodiments of the invention, the enzyme solution comprises 0.5-1 mmol/L Tris-HClpH8.0, such as 0.5-0.8 mmol/L or 0.8-1 mmol/L LTris-HCl pH8.0. According to certain embodiments of the invention, the enzyme solution comprises 0.5mmol/L, 0.6mmol/L, 0.7mmol/L, 0.8mmol/L, 0.9mmol/L or 1mmol/L of LTris-HCl pH 8.0.

According to certain embodiments of the invention, the enzyme solution comprises 2.5 to 2.7mmol/L KCl, such as 2.5 to 2.6mmol/L or 2.6 to 2.7 mmol/LKCl. According to certain embodiments of the invention, the enzyme solution comprises 2.5mmol/L, 2.51mmol/L, 2.52mmol/L, 2.53mmol/L, 2.54mmol/L, 2.55mmol/L, 2.56mmol/L, 2.57mmol/L, 2.58mmol/L, 2.59mmol/L, 2.6mmol/L, 2.61mmol/L, 2.62mmol/L, 2.63mmol/L, 2.64mmol/L, 2.65mmol/L, 2.66mmol/L, 2.67mmol/L, 2.68mmol/L, 2.69mmol/L or 2.7 mmol/LKCl.

According to some embodiments of the invention, the enzyme solution comprises 0.5 to 1mmol/L EDTA, such as 0.5 to 0.8mmol/L or 0.8 to 1mmol/L EDTA. According to certain embodiments of the invention, the enzyme solution comprises 0.5mmol/L, 0.6mmol/L, 0.7mmol/L, 0.8mmol/L, 0.9mmol/L or 1 mmol/LEDTA.

According to certain embodiments of the invention, the enzyme solution comprises 2% to 10% BSA, for example 2% to 8%, 2% to 5%, 5% to 10%, 5% to 8% or 8% to 10% BSA. According to certain embodiments of the invention, the enzyme solution comprises 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% BSA.

According to certain embodiments of the invention, the enzyme solution comprises 1000U/reaction M-MLV reverse transcriptase, 1500U/reaction T7RNA polymerase, 1mmol/L Tris-HCl pH8.0, 2.7mmol/L KCl, 1mmol/L EDTA, 10% BSA.

Lysis solution

Preferably, the lysis solution contains 10-20% Triton X-100, 2-10 mol/L guanidinium isothiocyanate, 2-10 mmol/L Tris HCl and pH 2.0-9.0.

According to certain embodiments of the invention, the lysate comprises 10% to 20% Triton X-100, such as 10% to 15% or 15% to 20% Triton X-100. According to certain embodiments of the invention, the lysate comprises 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% Triton X-100.

According to certain embodiments of the invention, the lysis solution comprises 2 to 10mol/L guanidinium isothiocyanate, such as 2 to 8mol/L, 2 to 5mol/L, 5 to 10mol/L, 5 to 8mol/L, or 8 to 10mol/L guanidinium isothiocyanate. According to certain embodiments of the invention, the lysis solution comprises 2mol/L, 3mol/L, 4mol/L, 5mol/L, 6mol/L, 7mol/L, 8mol/L, 9mol/L or 10mol/L guanidinium isothiocyanate.

According to certain embodiments of the invention, the lysis solution comprises 2-10 mmol/L Tris HCl, such as 4-10 mmol/L, 6-10 mmol/L, 8-10 mmol/L, 2-8 mmol/L, 4-8 mmol/L, 6-8 mmol/L, 2-6 mmol/L, 4-6 mmol/L or 2-4 mmol/L LTris HCl. According to certain embodiments of the invention, the lysis solution comprises 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10mmol/L of HCl.

According to some embodiments of the invention, the pH of the lysate is from pH2.0 to pH9.0, e.g., from pH2.0 to pH7.0, from pH2.0 to pH5.0, from pH5.0 to pH9.0, from pH5.0 to pH7.0, or from pH7.0 to pH 9.0. According to some embodiments of the invention, the pH of the lysate is pH2.0, pH3.0, pH4.0, pH5.0, pH6.0, pH7.0, pH8.0 or pH 9.0.

According to certain embodiments of the invention, the lysate comprises 10% Triton X-100, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH 8.0.

Nucleic acid extract

Preferably, the nucleic acid extracting solution contains 40-200 mM EDTA, 50-500 mg/L magnetic beads and 1-50 μ M capture probes.

According to some embodiments of the invention, the extract comprises 40-200 mM EDTA, such as 40-150 mM, 40-100 mM, 40-60 mM, 60-200 mM, 60-150 mM, 60-100 mM, 100-200 mM, 100-150 mM or 150-200 mM EDTA. According to certain embodiments of the invention, the extract comprises 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM or 200mM EDTA.

According to some embodiments of the present invention, the extract solution comprises 50-500 mg/L of magnetic beads, such as 50-400 mg/L, 50-300 mg/L, 50-200 mg/L, 50-100 mg/L, 100-500 mg/L, 100-400 mg/L, 100-300 mg/L, 100-200 mg/L, 200-500 mg/L, 200-400 mg/L, 200-300 mg/L, 300-500 mg/L, 300-400 mg/L, or 400-500 mg/L of magnetic beads. According to some embodiments of the invention, the extract comprises 50mg/L, 60mg/L, 70mg/L, 80mg/L, 90mg/L, 100mg/L, 130mg/L, 150mg/L, 180mg/L, 200mg/L, 230mg/L, 250mg/L, 280mg/L, 300mg/L, 330mg/L, 350mg/L, 380mg/L, 400mg/L, 430mg/L, 450mg/L, 480mg/L or 500mg/L of magnetic beads.

According to some embodiments of the present invention, the extract comprises 1-50 μ M capture probe, such as 1-40 μ M, 1-30 μ M, 1-20 μ M, 1-10 μ M, 10-50 μ M, 10-40 μ M, 10-30 μ M, 10-20 μ M, 20-50 μ M, 20-40 μ M, 20-30 μ M, 30-50 μ M, 30-40 μ M or 40-50 μ M capture probe. According to certain embodiments of the invention, the extract comprises 1 μ M, 5 μ M, 10 μ M, 15 μ M, 20 μ M, 25 μ M, 30 μ M, 35 μ M, 40 μ M, 45 μ M or 50 μ M capture probe.

According to some embodiments of the invention, the oligo (dT) is 10-26, such as 10-20, 10-18, 10-15, 10-12, 12-26, 12-20, 12-18, 12-15, 15-26, 15-20, 15-18, 18-26, 18-20, or 20-26 in length. According to some embodiments of the invention, the oligo (dT) is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 in length.

According to some embodiments of the invention, the extract comprises 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe.

According to some embodiments of the invention, the extract comprises 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe, which is oligo (dT).

According to some embodiments of the invention, the extract comprises 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe, the capture probe being oligo (dT), the oligo (dT) having a length of 18.

Cleaning solution

Preferably, the washing solution contains 2-2.5 mmol/L NaCl, 2-10 mol/L guanidinium isothiocyanate, 2-10 mmol/L Tris HCL, pH 7.0-9.0 and 2-10% SDS.

According to some embodiments of the invention, the washing solution comprises 2-2.5 mmol/L NaCl, such as 2-2.3 mmol/L or 2.3-2.5 mmol/L NaCl. According to certain embodiments of the invention, the wash solution comprises 2mmol/L, 2.1mmol/L, 2.2mmol/L, 2.3mmol/L, 2.4mmol/L or 2.5mmol/L NaCl.

According to some embodiments of the invention, the washing solution comprises 2 to 10mol/L of guanidinium isothiocyanate, such as 4 to 10mmol/L, 6 to 10mmol/L, 8 to 10mmol/L, 2 to 8mmol/L, 4 to 8mmol/L, 6 to 8mmol/L, 2 to 6mmol/L, 4 to 6mmol/L or 2 to 4mmol/L of guanidinium isothiocyanate. According to certain embodiments of the invention, the wash solution comprises 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10mmol/L guanidinium isothiocyanate.

According to certain embodiments of the invention, the washing solution comprises 2-10 mmol/L Tris HCl, such as 4-10 mmol/L, 6-10 mmol/L, 8-10 mmol/L, 2-8 mmol/L, 4-8 mmol/L, 6-8 mmol/L, 2-6 mmol/L, 4-6 mmol/L or 2-4 mmol/L LTris HCl. According to certain embodiments of the invention, the washing solution comprises 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10mmol/L of HCl.

According to certain embodiments of the invention, the pH of the wash solution is between pH7.0 and pH9.0, such as between pH7.0 and pH8.0 or between pH8.0 and pH 9.0. According to certain embodiments of the invention, the pH of the wash solution is pH7.0, pH7.5, pH8.0, pH8.5 or pH 9.0.

According to certain embodiments of the invention, the wash solution comprises 2% to 10% SDS, for example 2% to 8%, 2% to 5%, 5% to 10%, 5% to 8% or 8% to 10% SDS. According to certain embodiments of the invention, the wash solution comprises 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% SDS.

According to certain embodiments of the invention, the wash solution comprises 2mmol/L NaCl, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH8.0, 10% SDS.

Control

Preferably, the positive control comprises 10⁴～10⁶copies/mL Mycoplasma bovis RNA.

According to certain embodiments of the invention, the positive control comprises 10⁴～10⁶copies/mL Mycoplasma bovis RNA, e.g. 10⁴～10⁵copy/mL or 10⁵～10⁶copies/mL Mycoplasma bovis RNA. According to certain embodiments of the invention, the positive control comprises 10⁴copy/mL, 10⁵copy/mL or 10⁶copies/mL Mycoplasma bovis RNA.

Preferably, the negative control comprises physiological saline.

Further preferably, the kit comprises:

(1) lysis solution: 10-20% of Triton X-100, 2-10 mol/L of guanidinium isothiocyanate, 2-10 mmol/L of Tris HCl and pH 2.0-pH 9.0;

(2) nucleic acid extracting solution: 40-200 mM EDTA, 50-500 mg/L magnetic beads, 1-50 μ M capture probe;

(3) washing liquid: 2-2.5 mmol/L NaCl, 2-10 mol/L guanidinium isothiocyanate, 2-10 mmol/L LTris HCl, pH 7.0-9.0, 2-10% SDS;

(4) reaction solution: 2 to 10mmol/L Tris HCl, 20 to 40mmol/L MgCl₂，0.5～5mM dNTPs，1～10mM NTPs，2.5～2.7mmol/L KCl，2～10mmol/L Na₂HPO₄，0.5～2mmol/L KH₂PO₄；

(5) Detection liquid: 5-15 pmol/L of upstream primer MB nT7, 5-15 pmol/L of downstream primer MB T7, 5-10 pmol/LMB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/L of EDTA, and the balance of DEPC water;

(6) enzyme solution: 500-1000U/reaction M-MLV reverse transcriptase, 500-1500U/reaction T7RNA polymerase, 0.5-1 mmol/L Tris-HCl pH8.0, 2.5-2.7 mmol/L KCl, 0.5-1 mmol/L EDTA, 10% BSA;

(7) positive control: 10⁴～10⁶copy/mL mycoplasma bovis RNA;

(8) negative control: physiological saline.

Further preferably, the kit comprises:

(2) nucleic acid extracting solution: 40-200 mM EDTA, 50-500 mg/L magnetic beads, 1-50 μ M capture probe; the capture probe is oligo (dT);

(7) positive control: 10⁴～10⁶copy/mL mycoplasma bovis RNA;

(8) negative control: physiological saline.

Further preferably, the kit comprises:

(2) nucleic acid extracting solution: 40-200 mM EDTA, 50-500 mg/L magnetic beads, 1-50 μ M capture probe; the capture probe is oligo (dT), and the length of oligo (dT) is 10-26;

(5) Detection liquid: 5-15 pmol/L of upstream primer MB nT7, 5-15 pmol/L of downstream primer MBT7, 5-10 pmol/LMB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/L of EDTA, and the balance of DEPC water;

(7) positive control: 10⁴～10⁶copy/mL mycoplasma bovis RNA;

(8) negative control: physiological saline.

Further preferably, the kit comprises:

(1) lysis solution: 10% TritonX-100, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH 8.0;

(2) nucleic acid extracting solution: 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe;

(3) washing liquid: 2mmol/L NaCl, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH8.0, 10% SDS;

(4) reaction solution: 10mmol/L Tris-HCl, 40mmol/L MgCl₂，2.5mM dNTPs，6mM NTPs，2.7mmol/L KCl，10mmol/L Na₂HPO₄，2mmol/L KH₂PO₄；

(5) Detection liquid: 10pmol/L of upstream primer MB nT7, 10pmol/L of downstream primer MB T7, 8pmol/L of fluorescent Probe MB Probe, 1mmol/L of Tris-HCl pH8.0, 0.25mM EDTA, and the balance of DEPC water.

(6) Enzyme solution: 1000U/reaction M-MLV reverse transcriptase, 1500U/reaction T7RNA polymerase, 1mmol/L Tris-HCl pH8.0, 2.7mmol/L KCl, 1mmol/L EDTA, 10% BSA;

(7) MB positive control: comprises 10⁵copy/mL mycoplasma bovis RNA;

(8) MB negative control: physiological saline.

Further preferably, the kit comprises:

(2) nucleic acid extracting solution: 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe; the capture probe is oligo (dT);

(7) MB positive control: comprises 10⁵copy/mL mycoplasma bovis RNA;

(8) MB negative control: physiological saline.

Further preferably, the kit comprises:

(1) lysis solution: 10% TritonX-100, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH 8.0:

(2) nucleic acid extracting solution: 100mM EDTA, 250mg/L magnetic beads, 30. mu.M capture probe; the capture probe is oligo (dT), and the length of oligo (dT) is 18;

(7) MB positive control: comprises 10⁵copy/mL mycoplasma bovis RNA;

(8) MB negative control: physiological saline.

Detection method of mycoplasma bovis

(1) extracting target RNA in a sample to be detected;

(2) carrying out RNA isothermal amplification on target RNA in a sample to be detected by using the primers and the probe in the technical scheme;

And judging whether the target RNA of the mycoplasma bovis is contained or not according to the ct value. The ct value of 55 < ct < 60 is the gray area, i.e. critical value, and needs to be checked and confirmed again.

According to certain embodiments of the invention, where the probe comprises a FAM fluorescent label, the detection uses a FAM channel. According to certain embodiments of the invention, when the probe comprises a HEX fluorescent label, the detecting uses a HEX channel. According to some embodiments of the invention, where the probe comprises a VIC fluorescent label, the detection uses a VIC channel.

Preferably, the sample to be detected is food.

Preferably, the food product may be infected with mycoplasma bovis.

Preferably, the amplification reaction system is a mixture of a reaction solution and a detection solution.

Preferably, the reaction condition of the amplification is that the reaction is carried out for 40-60 min at 42 +/-1 ℃ in a fluorescent PCR instrument.

Preferably, the reaction conditions for the amplification are 42 ℃. + -. 1 ℃ in a fluorescent PCR instrument, for example 41-42 ℃ or 42-43 ℃. Preferably, the amplification reaction condition is a reaction at 41 ℃, 42 ℃ or 43 ℃ in a fluorescent PCR instrument.

Preferably, the reaction condition of the amplification is 40-60 min, such as 40-50 min or 50-60 min, in a fluorescent PCR instrument. Preferably, the amplification reaction condition is 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min or 60min of reaction in a fluorescent PCR instrument.

According to some embodiments of the present invention, the extracting the target RNA in the sample to be detected comprises lysing the thallus in the sample to be detected with the lysis solution to obtain a lysis solution containing the target RNA; and adding the nucleic acid extracting solution into the lysis solution to combine the capture probes on the magnetic beads with the target RNA, washing with the washing solution, and removing the nucleic acids which are not combined with the magnetic beads to obtain the target RNA.

According to some embodiments of the present invention, the isothermal amplification of the target RNA in the sample to be detected by using the primers and the probe comprises adding the target RNA extracted in step (1) into an amplification reaction solution comprising the reaction solution and the detection solution, performing a first incubation and a second incubation, wherein the first incubation is performed at 62 ℃ ± 2 ℃ for 5-10 min, the second incubation is performed at 40 ± 1 ℃ for 5-10 min, then adding the enzyme solution, reacting at 42 ℃ ± 1 ℃ for 40-60 min, and synchronously recording the change of the fluorescence signal by using the detector.

Preferably, the detection method of mycoplasma bovis is performed by the kit according to the above technical scheme, and the specific operation method comprises:

(1) cracking thalli in a sample to be detected by using the lysis solution to obtain a cracking solution containing target RNA, adding the nucleic acid extracting solution into the cracking solution to combine the capture probe on the magnetic bead with the target RNA, washing by using the washing solution, and removing nucleic acid which is not combined with the magnetic bead to obtain the target RNA;

According to some embodiments of the invention, the target RNA extracted in step (1) is added to an amplification reaction mixture consisting of the reaction mixture and the detection mixture, and the first incubation is an incubation at 62 ℃. + -. 2 ℃, for example, 60-62 ℃ or 62-64 ℃. According to some embodiments of the present invention, (2) the target RNA extracted in step (1) is added to an amplification reaction solution composed of the reaction solution and the detection solution, and the first incubation is performed at 60 ℃, 61 ℃, 62 ℃, 63 ℃ or 64 ℃.

According to certain embodiments of the present invention, the target RNA extracted in step (2) is added to an amplification reaction solution composed of the reaction solution and the detection solution, and the first incubation is 5-10 min, such as 5-8 min or 8-10 min. According to some embodiments of the present invention, the target RNA extracted in step (1) is added to an amplification reaction solution consisting of the reaction solution and the detection solution, and the first incubation is 5min, 6min, 7min, 8min, 9min or 10 min.

According to some embodiments of the invention, the target RNA extracted in step (1) is added to an amplification reaction mixture consisting of the reaction mixture and the detection mixture, and the second incubation is at 40. + -. 1 ℃, for example 39-40 ℃ or 40-41 ℃. According to some embodiments of the present invention, (2) the target RNA extracted in step (1) is added to an amplification reaction solution consisting of the reaction solution and the detection solution, and the second incubation is at 39 ℃, 40 ℃ or 43 ℃.

According to some embodiments of the present invention, the target RNA extracted in step (1) is added to the amplification reaction solution composed of the reaction solution and the detection solution, and the second incubation is 5-10 min, such as 5-8 min or 8-10 min. According to some embodiments of the present invention, the target RNA extracted in step (1) is added to an amplification reaction solution consisting of the reaction solution and the detection solution, and the second incubation is 5min, 6min, 7min, 8min, 9min or 10 min.

According to some embodiments of the present invention, (2) the target RNA extracted in step (1) is added to an amplification reaction solution composed of the reaction solution and the detection solution, and the enzyme solution is added to the amplification reaction solution to perform a reaction at 42 ℃. + -. 1 ℃, for example, at 41 to 42 ℃ or 42 to 43 ℃. According to some embodiments of the present invention, (2) the target RNA extracted in step (1) is added to an amplification reaction solution composed of the reaction solution and the detection solution, and the enzyme solution is added to the amplification reaction solution to perform a reaction at 41 ℃, 42 ℃ or 43 ℃.

According to some embodiments of the present invention, the sample of ct ≦ 55 in (3) is positive, e.g., the samples of ct ≦ 54, ct ≦ 53, ct ≦ 52, ct ≦ 51, ct ≦ 50, ct ≦ 45, ct ≦ 40, ct ≦ 35, ct ≦ 30, ct ≦ 25, ct ≦ 20, ct ≦ 15, ct ≦ 10, ct ≦ 5 are positive. According to some embodiments of the invention, the sample with ct ≦ 55 in (4) is positive, e.g., the sample with ct being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 is positive.

According to some embodiments of the present invention, the sample of 55 < ct < 60 in (3) is suggested for retesting, for example, the sample of 55 < ct < 59, 55 < ct < 58, 55 < ct < 57, 55 < ct < 56, 56 < ct < 60, 56 < ct < 59, 56 < ct < 58, 56 < ct < 57, 57 < ct < 60, 57 < ct < 59, 57 < ct < 58, 58 < ct < 60, 58 < ct < 59, 59 < ct < 60. According to some embodiments of the invention, the sample in (3) with ct 56, 57, 58 or 59 is proposed for re-detection.

According to some embodiments of the invention, the sample with ct < 60 in the (3) review result is positive, for example, a sample with ct < 59, ct < 58, ct < 57, ct < 56, ct < 55, ct < 54, ct < 53, ct < 52, ct < 51, ct < 50, ct < 45, ct < 40, ct < 35, ct < 30, ct < 25, ct < 20, ct < 15, ct < 10, or ct < 5 is positive. According to some embodiments of the invention, the sample with ct < 60 in the (4) retest result is positive, for example, the sample with ct of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59 is positive.

According to some embodiments of the invention, the test sample is tested and evaluated with reference to the positive control and negative control results.

According to some embodiments of the invention, a positive test result indicates that there is viable mycoplasma bovis in the sample; a negative test result indicates that there is no viable Mycoplasma bovis in the sample.

The invention has the beneficial effects that:

the invention provides a primer, a probe and a kit for RNA isothermal amplification detection of mycoplasma bovis and a method for detecting mycoplasma bovis, and compared with the existing MB detection, the invention has the following advantages:

(1) high specificity: the specific fluorescent probe designed aiming at the MB target nucleic acid can efficiently and specifically detect the RNA of the MB;

(2) high sensitivity: the invention uses RNA as a template for amplification, the concentration of the RNA on the substrate of the strain is 1000 times of that of DNA, and the sensitivity can reach 10²copy/mL;

(3) and (3) rapid detection: the invention synchronously performs amplification and detection of nucleic acid, has high amplification efficiency, performs amplification at a copy/cycle rate of 100-1000 copies/cycle, and can amplify the template by 10 in 15-30 minutes⁹And (4) doubling. And the whole process is constant temperature, so the required time is greatly shortened, the amplification detection only needs 50min, the result can be obtained after the amplification detection is carried out for 150min, and the time is greatly shortened compared with the time of a national standard method.

(4) Can effectively distinguish dead bacteria and live bacteria in a detected object, has more accurate and scientific detection and avoids false positive.

(5) Pollution is easy to control: compared with real-time fluorescent PCR, the amplification product of the invention is RNA which is easy to degrade in nature, so that the pollution control is easier.

(6) The equipment is simple, and the cost is low: compared with real-time fluorescent quantitative PCR, the instrument used in the invention does not need temperature rise and fall circulation, thereby greatly reducing the design and production cost.

Therefore, the kit can detect the MB RNA in the food sample, and has high specificity and high sensitivity (up to 10)²copy/mL), accurate (avoid false positive) detection, low pollution (the amplified product RNA is easy to degrade in natural environment) and rapid detection (the amplification detection is completed in 150 min), and the method plays an important role in rapid detection of mycoplasma bovis and has a wide application prospect.

Description of the drawings:

FIG. 1 shows the results of sensitivity detection in example 3 of the present invention;

FIG. 2 shows the results of the specificity test in example 4 of the present invention;

FIG. 3 shows the results of testing food samples according to example 5 of the present invention; and

FIG. 4 shows the results of detection of MB target RNA after sterilization in example 6 of the present invention.

Detailed Description

The invention provides a primer and a probe for detecting mycoplasma bovis by RNA isothermal amplification in the technical scheme, wherein the primer and the probe are both specific sequences and can specifically detect mycoplasma bovis.

The sequences of the primers are respectively as follows:

the upstream primer was MB nT7 (primer containing no promoter): CTGACCTTCGGGTAAAGC, respectively; or TGGTTTGTCGCGTTGTTC;

the downstream primer was MB T7 (primer containing T7 promoter): AATTTAATAC AGCTCACTAT AGGGAGACCACACCGCT AGAGC, respectively; or AATTTAATAC GACTCACTAT AGGGAGAACCAGGAATTCCAGT; wherein, the downstream primer has a promoter sequence AATTTAATAC AGCTCACTAT AGGGAGA recognized by T7RNA polymerase.

In the invention, the MB fluorescent probe is a molecular beacon, is a hairpin structure with a ring and a self-complementary stem, and consists of single-stranded nucleic acid molecules with two ends respectively and covalently labeled with a fluorescent dye and a quencher, wherein the ring part of the molecular beacon is complementary with a target, and two ends are complementary to form the stem.

The nucleic acid molecule sequence of the probe is as follows: CUGCGGGACCCAGGGAUGCAUGUCGCAG, respectively; or CTGCGAGCGTG CGGGCGATAC GGG CAG CGCAG.

The 5 'end of the MB fluorescent probe is marked by FAM fluorescent group, and the 3' end is marked by DABCYL quenching group.

When the probe encounters the target RNA, the probe preferentially binds to the target RNA rather than forming a hairpin structure, and the stem structure is broken, the fluorescent reporter and the quencher are separated, and the reporter is allowed to fluoresce. The probe can be stably combined with a fluorescent agent and a quenching agent in the absence of the target RNA, and does not emit fluorescence, so that the background is low.

The invention provides a kit for detecting mycoplasma bovis by RNA isothermal amplification, which comprises reaction liquid, detection liquid containing the primers and the probes in the technical scheme, enzyme liquid containing T7RNA polymerase and M-MLV reverse transcriptase, lysis solution, nucleic acid extracting solution, washing liquid, positive control and negative control.

Preferably, the kit for detecting mycoplasma bovis by RNA isothermal amplification comprises the following reagents:

(1) lysis solution: 10-20% of Triton X-100, 4.5-5 mol/L of guanidinium isothiocyanate, 2-10 mmol/L of Tris HCl and pH 7.0-9.0; more preferably the lysis solution is: 10% Triton X-100, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH 8.0;

(2) nucleic acid extracting solution: 40-200 mM EDTA, 50-500 mg/L magnetic beads, 1-50 μ M capture probe; more preferably, the nucleic acid extract is: 150mM EDTA, 400mg/L magnetic beads, 40. mu.M capture probe;

(3) washing liquid: 2-2.5 mmol/L NaCl, 4.5-5 mol/L guanidinium isothiocyanate, 10mmol/L TrisHCl, pH8.0 and 10% SDS; more preferably, the washing solution is: 2mmol/L NaCl, 5mol/L guanidinium isothiocyanate, 10mmol/L TrisHCl, pH8.0, 10% SDS;

(4) reaction solution: 2 to 10mmol/L Tris HCl, 10 to 40mmol/L MgCl₂，0.5～5mM dNTPs，1～10mM NTPs，2.5～2.7mmol/L KCl，2～10mmol/L Na₂HPO₄，0.5～2mmol/L KH₂PO₄(ii) a More preferably, the reaction solution is 10mmol/L Tris HCl, 40mmol/L MgCl₂，2.5mM dNTPs，5mM NTPs，2.7mmol/L KCl，10mmol/L Na₂HPO₄，2mmol/L KH₂PO₄；

(5) Detection liquid: 5-15 pmol/L of upstream primer MB nT7, 5-15 pmol/L of downstream primer MB T7, 5-10 pmol/LMB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/L of EDTA, and the balance of DEPC water; more preferably, the detection solution is: 10pmol/L of upstream primer MB nT7, 10pmol/L of downstream primer MB T7, 10pmol/LMB fluorescent probe, 1mmol/L Tris-HCl pH8.0, 0.25mM EDTA, DEPC water;

(6) enzyme solution: 500-1000U/reaction M-MLV reverse transcriptase, 500-1500U/reaction T7RNA polymerase, 0.5-1 mmol/L Tris-HCl pH8.0, 2.5-2.7 mmol/L KCl, 0.5-1 mmol/L EDTA, 2-10% BSA; more preferably, the enzyme solution is: 500U/reaction M-MLV reverse transcriptase, 500U/reaction T7RNA polymerase, 1mmol/L Tris-HCl pH8.0, 2.7mmol/L KCl, 1mmol/L EDTA.10% BSA;

(7) positive control: mycoplasma bovis RNA;

(8) negative control: physiological saline.

Wherein the positive control preferably contains 10M M.bovis⁴～10⁶copies/mL of Mycoplasma bovis RNA. The reagents in the kit are conventional in the field, and the source of the reagents is not limited in the invention and can be purchased commercially.

The kit for detecting mycoplasma bovis adopts an RNA isothermal amplification detection method, and uses M-MLV reverse transcriptase and T7RNA polymerase to simultaneously realize nucleic acid amplification, wherein the M-MLV reverse transcriptase is used for generating one DNA copy of target RNA, the T7RNA polymerase generates a plurality of RNA copies from the DNA copy, a probe with a fluorescent label is specifically combined with the amplified target RNA to generate fluorescence, and the fluorescent signal can be captured by a detection instrument. The kit can detect RNA in mycoplasma bovis, and has the characteristics of high specificity, high sensitivity and low pollution (amplification product RNA is easy to degrade in natural environment).

The invention also provides a detection method of mycoplasma bovis, which comprises the following steps:

(1) extracting target RNA in a sample to be detected;

(2) RNA isothermal amplification is carried out on RNA of thalli in a sample to be detected by using the primer and the probe of any one of the technical schemes, the ct value of the reaction of the sample to be detected is recorded, and whether the target RNA of the mycoplasma bovis is contained is judged according to the ct value.

In the present invention, the sample to be detected is preferably cow milk, the source of the cow milk is not particularly limited, and cow milk prepared or stored at will or cow milk of any brand commercially available can be detected. In the present invention, preferably, a sample to be tested is dissolved in physiological saline, and after the thalli are lysed, RNA of the thalli is extracted as a template for isothermal amplification. The reagent for lysing bacteria and extracting RNA according to the present invention is preferably used in the kit according to the above-mentioned embodiment of the present invention, and the methods are well known to those skilled in the art.

The amplification reaction can be optionally carried out in a fluorescent PCR instrument, the reaction temperature is preferably 40-44 ℃, more preferably 42 ℃, and the reaction time is preferably 40-60 min, more preferably 50 min.

In the present invention, the detection method of mycoplasma bovis is preferably performed by the kit according to any one of the above technical solutions, and the specific operation method includes:

(2) adding the target RNA extracted in the step (1) into an amplification reaction solution consisting of the reaction solution and the detection solution, incubating for 10min at 60 +/-0.1 ℃, then incubating for 5min at 42 +/-0.1 ℃, then adding the enzyme solution, starting to react for 50min at 42 +/-0.1 ℃, and synchronously recording the change of a fluorescence signal by using a detector;

(3) according to the time and the intensity of the fluorescent signal, detecting and judging the sample to be detected by referring to the MB positive control result and the MB negative control result; the judging method comprises the following steps: if the detection result is positive, the sample contains live mycoplasma bovis, and if the detection result is negative, the sample does not contain live mycoplasma bovis.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1: design of special primer and probe for RNA isothermal amplification detection of Mycoplasma Bovis (MB)

In this example, an upstream primer MB nT7, a downstream primer MB T7 and an MB detection probe for RNA isothermal amplification detection of Mycoplasma Bovis (MB) were designed.

The invention selects MB bacteria highly conserved segment 16s rRNASEQ ID NO: 1 as an amplification target sequence region, and manually designing special primers and probe sequences for real-time fluorescent nucleic acid isothermal amplification detection of Mycoplasma Bovis (MB) by using DNAman software according to the design principle of the primers and the probes to obtain the following specific sequences:

(1) a pair of MB detection primers for generating a DNA copy of MB target nucleic acid (MB RNA) under the action of M-MLV reverse transcriptase, wherein the MB detection primers consist of an upstream primer MB nT7 and a downstream primer MB T7, the upstream primer MB nT7 primer sequence is 5'-CTTACCAGGT GTTGACATCC-3' (SEQ ID NO: 2), and the downstream primer MB T7 primer sequence is 5'-AATTTAATACGACTCACTATAGGGAGACAACATCTCAGAACACGAGCT-3' (SEQ ID NO: 3);

(2) an MB detection Probe MB Probe for specific binding based on RNA copies generated by DNA copies of the MB target nucleic acid (MB RNA) under the action of T7RNA polymerase, the nucleotide sequence of the MB detection Probe MB Probe is 5'-CACCGAGTGC CTTCGGGAACGGTG-3', (SEQ ID NO: 4), the 5 'end is fluorescently labeled with FAM, and the 3' end is fluorescently labeled with DABCYL.

Example 2: RNA isothermal amplification detection kit for preparing Mycoplasma Bovis (MB)

This example prepares a kit for isothermal amplification of RNA for detection of Mycoplasma Bovis (MB).

The RNA isothermal amplification detection kit for Mycoplasma Bovis (MB) of the present invention was obtained using the primers and probes provided in example 1. The kit comprises a downstream MBT7 primer, an upstream MBnT7 primer, an MB detection probe, M-MLV reverse transcriptase and T7RNA polymerase.

The capture probe exists in a nucleic acid extracting solution, the downstream MBT7 primer, the upstream MBnT7 primer and the MB detection probe exist in an MB detection solution, and the M-MLV reverse transcriptase and the T7RNA polymerase exist in an enzyme solution; specifically, the main components of the kit are as follows:

(1) lysis solution: 10% Triton X-100, 5mol/L guanidinium isothiocyanate, 10mmol/L Tris HCl, pH 8.0;

(4) MB reaction solution: 10mmol/L Tris-HCl, 40mmol/L MgCl₂，2.5mM dNTPs，6mM NTPs，2.7mmol/L KCl，10mmol/L Na₂HPO₄，2mmol/L KH₂PO₄；

(5) MB detection solution: 10pmol/L of upstream primer MB nT7, 10pmol/L of downstream primer MB T7, 8pmol/L of fluorescent Probe MB Probe, 1mmol/L of Tris-HCl pH8.0, 0.25mM EDTA, DEPC water.

(7) MB positive control: comprises 10⁵copy/mL mycoplasma bovis RNA;

(8) MB negative control: physiological saline.

Example 3: cattle branchSensitivity of RNA isothermal amplification detection of protomer

This example shows the sensitivity of isothermal amplification of RNA for detection of Mycoplasma bovis.

The kit (composition is shown in example 2) of the invention is used for detecting Mycoplasma Bovis (MB), and the specific method comprises the following steps:

(1) target RNA dilution

Measured at a concentration of 10⁸copies/mL of Mycoplasma bovis RNA, 10-fold gradient diluted to 10⁷copy/mL, 10⁶copy/mL, 10⁵copy/mL, 10⁴copy/mL, 10³copy/mL, 10²copy/mL, 10¹copy/mL, 10⁰copy/mL.

(2) Nucleic acid extraction

To a 1.5mL EP tube, 200. mu.L of lysate, 100. mu.L of nucleic acid extract, and 200. mu.L of target RNA were added, mixed well, incubated at 60 ℃ for 10 minutes, and allowed to stand at room temperature for 10 minutes. The centrifuge tube was placed on a magnetic bead separator and allowed to stand for 5 minutes. Keeping the centrifugal tube on a magnetic bead separator, sucking liquid and keeping magnetic beads; adding 1ml of washing solution, shaking and uniformly mixing, then placing the mixture back to the magnetic bead separator, repeatedly washing once, and removing the sample processing tube from the magnetic bead separator to obtain a magnetic bead-nucleic acid compound in the tube for later use.

(3) Isothermal amplification assay

To the sample processing tube, 40. mu.L of a reaction detection solution (40. mu. LMB reaction solution + 2.5. mu. LMB detection solution) was added, and the concentration of the downstream MBT7 primer, the concentration of the upstream MBnT7 primer and the concentration of the MB detection probe in the MB detection solution were 10pmol, 10pmol and 5pmol, respectively.

Adding 30 mu L of the reaction detection solution which is uniformly mixed by oscillation into a micro reaction tube, preserving the heat at 60 ℃ for 10 minutes, and preserving the heat at 42 ℃ for 5 minutes; mu.L of the enzyme solution preheated to 42 ℃ was added to the micro reaction tube, and the mixture was shaken at 1200rpm for 15 seconds and mixed.

The micro reaction tube was transferred to the channel IV of a Tianlong TL988 PCR apparatus (product of Sainan Tianlong Co.), reacted at 42 ℃ for 50 minutes, and fluorescence was detected every 50 seconds for 60 times. It will be appreciated by those skilled in the art that a FAM channel PCR instrument can be used to perform the present experiment.

(4) Determination of results

And setting a threshold line according to a curve obtained by the PCR amplification result, reading the ct value, and judging the result.

Setting a threshold value: the threshold line is just above the highest point of the normal negative control amplification curve. ct represents the abscissa reading of the intersection of the sample curve and the threshold line;

(1) and (3) judging a positive result:

FAM channel: the sample with ct less than or equal to 55 is positive; samples 55 < ct < 60 are recommended for retesting, and the retest result is FAM channel: samples with ct < 60 were positive.

(2) And (4) judging a negative result: FAM channel ct has no value or is 60.

(5) Results

FIG. 1 shows a sensitivity test chart in which the concentration of MB strain RNA is 10²copy/mL, the detection result is positive, namely the lower limit sensitivity of the detection can reach 10²copy/mL, high sensitivity, and higher recognition degree for low-concentration polluted samples, so the kit is safer and more time-saving for quality control detection.

Example 4: RNA isothermal amplification detection specificity of mycoplasma bovis

This example detects the specificity of isothermal amplification of RNA for detection of Mycoplasma bovis.

This detection mode is another application of the present invention: the composition of the kit was the same as in example 2.

The specific reference sample treatment method is as follows: the 11 negative reference samples included salmonella, salmonella typhimurium, vibrio parahaemolyticus, escherichia coli O157: h7, Proteus vulgaris, Citrobacter, Bacillus cereus, Listeria monocytogenes, Shigella, Staphylococcus aureus, and Klebsiella. A positive control (Mycoplasma bovis RNA) was also included.

The detection method is the same as that of example 3, and the amount of the reagent used in the detection is the same as that of example 3.

The detection results using the kit of the present invention are shown in FIG. 3, and the amplification curves of 11 negative reference samples do not cross the threshold line, and thus the sample can be clearly determined to be negative.

FIG. 2 shows that the kit of the present invention has good specificity to Mycoplasma bovis, and can accurately detect Mycoplasma bovis without being interfered by other bacteria.

Example 5: RNA isothermal amplification detection of real food samples

This example detects isothermal amplification of RNA from real food samples.

This detection mode is another application of the present invention: the composition of the kit is the same as that in example 2, the detection method is the same as that in example 3, the amount of the reagent used in the detection is the same as that in example 3, and the type of the food sample for detecting mycoplasma bovis comprises the following steps: the specific method of the cow milk comprises the following steps:

(1) food sample treatment:

100mL of the sample is put into a sterilized conical flask, 900mL of physiological saline is added, and the mixture is fully and uniformly mixed. The mycoplasma bovis sample number is MB food sample 1-5, the No. 5 sample is fresh cow milk of different storefronts of Zhang Jiakou city, and positive control (containing 10)⁵copies/mL mycoplasma bovis RNA), negative controls (saline), one each.

(2) Nucleic acid extraction

The specific procedure was the same as in example 3.

(3) Isothermal amplification assay

The specific procedure was the same as in example 3.

(4) And (4) judging a result:

(1) and (3) judging a positive result:

f1 channel (FAM channel): the sample with ct less than or equal to 55 is positive; samples 55 < ct < 60 are recommended for retesting, test result F1 channel: samples with ct < 60 were positive.

(2) And (4) judging a negative result: f1 channel ct has no value or is negative at 60.

Quality control: positive and negative controls were set for each test, and the results should simultaneously satisfy the positive control F1 channel: ct is less than or equal to 55; negative control F1 channel: ct has no value or is 60, otherwise, the detection result is regarded as invalid.

(5) Results

According to the ct value of the F1 channel, the samples were judged to be negative, and the result is shown in FIG. 3, which shows that the method of the present invention can be used for detecting whether commercially available cow milk contains mycoplasma bovis.

Example 6: comparison of viable and dead bacteria in RNA isothermal amplification detection

This example compares the isothermal amplification of RNA to detect viable and dead bacteria.

This test is another application of the present invention: the composition of the kit, the detection method and the reagent dosage are the same as those in the embodiment 3, and the specific method comprises the following steps:

(1) sample processing

Taking target RNA for detecting mycoplasma bovis (10)⁸copy/mL), diluted to 10 with physiological saline, respectively⁷copy/mL, 10⁶copy/mL, 10⁵copy/mL, 10⁴copy/mL, 10³copy/mL, 10²copy/mL, autoclaved at 121 ℃ for 30 minutes, and then placed at normal temperature for 12 hours.

(2) Nucleic acid extraction

The concrete operation steps are the same as in example 3

(3) Isothermal amplification assay

The constant temperature detection method was the same as in example 3.

(4) The result of the detection

Detecting each gradient sample, and detecting 10 by the method⁸copy/mL, 10⁷copy/mL, 10⁶copy/mL, and 10⁵copy/mL, 10⁴copy/mL, 10³copy/mL, 10²copy/mL was not detected (FIG. 4), and the amount of dead bacteria RNA in the actual sample was less than 10⁶copy/mL, so this experiment can show that after the cell death, its RNA is also degraded, so in the above-mentioned limited concentration as long as there is no viable bacteria this method can not be detected, it can be seen that the invention can distinguish dead viable bacteria, eliminate the result that the false positive appears, also have higher sensitivity.

The SAT method is firstly used for detecting mycoplasma bovis, and the method has high sensitivity and good specificity after experiments, and the RNA concentration of the bacterial liquid is 10⁶Viable and dead bacteria can be distinguished under copy/mL, a detection method based on RNA as a template is provided, and a new detection method for the detection industry are provided(ii) a pathway.

Based on the disclosure of the present invention, those skilled in the art can implement the isothermal amplification detection kit for Mycoplasma Bovis (MB) RNA claimed in the present invention without undue experimentation, and achieve the desired effect. The disclosed embodiments of the present invention are merely detailed descriptions of the present invention and are not to be construed as limiting the present invention. It will be apparent to those skilled in the art that similar substitutes or modifications for obvious substitutions or modifications of the invention, or certain chemically or biologically structurally and functionally related agents may be substituted for the agents described herein or modifications may be made thereto without departing from the spirit, scope and concept of the invention and within the scope and ambit of the appended claims.

Sequence listing

<110> Zhang Jiakou Jianyuan science and technology Limited

<120> primer, probe, kit and detection method for RNA isothermal amplification detection of mycoplasma bovis

<130>TQZX2019-ZL1606

<141>2019-12-19

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<170>SIPOSequenceListing 1.0

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ttttgtttgg agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg 60

caagtcgaac ggaaaggtct cttcggagat actcgagtgg cgaacgggtg agtaacacgt 120

gggtgatctg ccctgcactt cgggataagc ctgggaaact gggtctaata ccggatagga 180

ccacgggatg catgtcttgt ggtggaaagc gctttagcgg tgtgggatga gcccgcggcc 240

tatcagcttg ttggtggggt gacggcctac caaggcgacg acgggtagcc ggcctgagag 300

ggtgtccggc cacactggga ctgagatacg gcccagactc ctacgggagg cagcagtggg 360

gaatattgca caatgggcgc aagcctgatg cagcgacgcc gcgtggggga tgacggcctt 420

cgggttgtaa acctctttca ccatcgacga aggtccgggt tctctcggat tgacggtagg 480

tggagaagaa gcaccggcca actacgtgcc agcagccgcg gtaatacgta gggtgcgagc 540

gttgtccgga attactgggc gtaaagagct cgtaggtggt ttgtcgcgtt gttcgtgaaa 600

tctcacggct taactgtgag cgtgcgggcg atacgggcag actagagtac tgcaggggag 660

actggaattc ctggtgtagc ggtggaatgc gcagatatca ggaggaacac cggtggcgaa 720

ggcgggtctc tgggcagtaa ctgacgctga ggagcgaaag cgtggggagc gaacaggatt 780

agataccctg gtagtccacg ccgtaaacgg tgggtactag gtgtgggttt ccttccttgg 840

gatccgtgcc gtagctaacg cattaagtac cccgcctggg gagtacggcc gcaaggctaa 900

aactcaaagg aattgacggg ggcccgcaca agcggcggag catgtggatt aattcgatgc 960

aacgcgaaga accttacctg ggtttgacat gcacaggacg cgtctagaga taggcgttcc 1020

cttgtggcct gtgtgcaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1080

ttaagtcccg caacgagcgc aacccttgtc tcatgttgcc agcacgtaat ggtggggact 1140

cgtgagagac tgccggggtc aactcggagg aaggtgggga tgacgtcaag tcatcatgcc 1200

ccttatgtcc agggcttcac acatgctaca atggccggta caaagggctg cgatgccgcg 1260

aggttaagcg aatccttaaa agccggtctc agttcggatc ggggtctgca actcgacccc 1320

gtgaagtcgg agtcgctagt aatcgcagat cagcaacgct gcggtgaata cgttcccggg 1380

ccttgtacac accgcccgtc acgtcatgaa agtcggtaac acccgaagcc agtggcctaa 1440

ccctcgggag ggagctgtcg aaggtgggat cggcgattgg gacgaagtcg taacaaggta 1500

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ctgcacttcg ggataagc 18

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aatttaatac gactcactat agggagacca caccgctaaa cg 42

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Claims

1. A primer and a probe for RNA isothermal amplification detection of mycoplasma bovis,

the primer comprises:

upstream primer MB nT 7: the upstream primer MB nT7 comprises: CTGACCTTCGGGTAAAGC, or TGGTTTGTCGCGTTGTTC, or a homologous sequence thereof;

the downstream primer MB T7: the downstream primer MB T7 comprises a T7 promoter sequence and a target primer sequence; wherein the target primer sequence comprises at least 8 nucleotide sequences of CCACACCGCT AGAGC, or ACCAGGAATTCCAGT, or homologous sequences thereof; the T7 promoter sequence comprises at least 10 nucleotide sequences of AATTTAATAC AGCTCACTAT AGGGAGA, or a homologous sequence thereof;

the probe has a hairpin structure with a loop and a stem that is self-complementary.

2. The primer and probe according to claim 1, wherein the probe is covalently labeled with a fluorophore and a quencher at both ends thereof, respectively, preferably the fluorophore comprises FAM, HEX or VIC, and preferably the quencher comprises DABCYL.

3. The primers and probe of claim 1, wherein the probe sequence comprises at least 10 nucleotides of CUGCGGGACCCAGGGAUGCAUGUCGCAG, or CTGCG AGCGTG CGGGCGATAC GGG CAG CGCAG, or a homologous sequence thereof.

4. The primers and probes according to claim 1 or 3, wherein said homologous sequence refers to a nucleotide sequence which is at least 70% identical to said sequence, preferably said homologous sequence has substantially the same activity as the sequence disclosed herein.

5. A kit for detecting mycoplasma bovis by RNA isothermal amplification comprises a reaction solution, a detection solution containing the primers and the probes, and an enzyme solution containing T7RNA polymerase and M-MLV reverse transcriptase.

6. The kit of claim 5, wherein the kit comprises:

Detection liquid: the detection solution comprises 5-15 pmol/L of an upstream primer MB nT7, 5-15 pmol/L of a downstream primer MB T7, 5-10 pmol/L of an MB fluorescent probe, 1-5 mmol/L of Tris-HCl pH8.0, 1-5 mmol/L EDTA and the balance of DEPC water; and

7. The kit according to claim 5 or 6, wherein the kit further comprises a lysis solution, a nucleic acid extraction solution, a washing solution, a positive control and a negative control.

8. The kit of claim 7, wherein the kit further comprises:

negative control: the negative control comprises saline.

9. A method for detecting Mycoplasma bovis using the kit of any one of claims 5-8, wherein said method comprises the steps of:

(1) extracting target RNA in a sample to be detected;

10. The method of claim 9, the method comprising:

(2) adding the target RNA extracted in the step (1) into an amplification reaction solution consisting of the reaction solution and a detection solution, performing first incubation and second incubation, wherein the first incubation is performed at 62 +/-2 ℃ for 5-10 min, the second incubation is performed at 40 +/-1 ℃ for 5-10 min, then adding the enzyme solution, reacting at 42 +/-1 ℃ for 40-60 min, and synchronously recording the change of a fluorescence signal by using a detector;