CN101481742A - Detection kit for Mycoplasma hyopneumoniae and use thereof - Google Patents

Abstract

The invention discloses a detection kit for mycoplasma hyopneumoniae and its application. The invention provides special primers for detecting Mycoplasma hyopneumoniae, which consist of the following three pairs of primers used for loop-mediated isothermal amplification: a pair of primers is an inner primer pair combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360 , a pair of primers is a pair of outer primers combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360, and a pair of primers is a pair of circular primers combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360. The Mycoplasma hyopneumoniae detection kit provided by the invention is a kit including the special primers. The detection kit for mycoplasma hyopneumoniae of the present invention has high detection sensitivity, can detect target DNA with 10 copies, is simple and convenient to operate, and is especially suitable for clinical medical detection at grassroots sites and detection of mycoplasma hyopneumoniae that may be contaminated in food.

Description

A kind of mycoplasma hyopneumoniae detection kit and its application

technical field

The invention relates to a detection kit for mycoplasma hyopneumoniae and its application.

Background technique

Mycoplasma swine pneumonia is a chronic respiratory infectious disease with high morbidity and low mortality in pigs caused by Mycoplasma hyopneumoniae. It is often called swine asthma (or panting disease) in my country. Mycoplasma swine pneumonia is widely distributed all over the world, and the infection rate in pig herds is 30%-80%. At present, most pig raising countries have reported the occurrence of this disease. After the onset, chronic dry cough may occur, long-term growth and development are poor, growth rate and feed conversion rate are reduced, and the general mortality rate is not high, but secondary infection can also cause serious death, resulting in great economic losses, which are harmful to the development of pig industry. It is one of the most frequently occurring and economically significant pig diseases.

The existing detection methods of Mycoplasma hyopneumoniae can be roughly divided into two categories, one is the traditional pathogen isolation and culture method, and the other is the molecular biology method. Since M. hyopneumoniae is one of the most difficult pathogens to isolate and identify, it grows slowly, takes a long time, and is often masked by M. hyorhinosus overgrowth, and it can be difficult to isolate hyopneumoniae from drug-treated or recovered pigs Mycoplasma, so pathogen isolation and culture are not often used in clinical testing; molecular biology methods mainly include polymerase chain reaction (PCR) and nucleic acid probe detection. PCR detection also includes conventional PCR, random primer PCR, nested PCR, RealTime PCR and other methods. The PCR method is fast and sensitive, but requires the use of special instruments, such as PCR machines, and the preparation of fluorescent probes is relatively expensive, which is not suitable for grassroots applications.

Contents of the invention

The object of the present invention is to provide a kit for rapid detection of Mycoplasma hyopneumoniae based on loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technology.

The present invention provides special primers for detecting Mycoplasma hyopneumoniae, which are composed of the following three pairs of primers used for loop-mediated isothermal amplification: a pair of primers is an inner primer pair combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360 A pair of primers is a pair of outer primers combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360, and a pair of primers is a pair of circular primers combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360.

Specifically, among the special primers, a pair of primers may be able to bind to the nucleotide region from 876699 to 876919 at the 5' end of the Mycoplasma hyopneumoniae genome GenBank Accession Number NC-006360 (16S ribosomal RNA gene partial sequence) A pair of inner primers, a pair of primers can be combined in the Mycoplasma hyopneumoniae genome GenBank Accession Number NC-006360 from the 5' end 876699 to 876919 nucleotide region (16S ribosomal RNA gene partial sequence) outer primer pair, A pair of primers can be a pair of circular primers that can bind to the nucleotide region from 876699 to 876919 at the 5' end (16S ribosomal RNA gene partial sequence) in GenBank Accession Number NC-006360 of the Mycoplasma hyopneumoniae genome.

More specifically, the inner primer pair includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer can be sequence 4 in the sequence listing, and the nucleotide sequence of the downstream primer can be sequence 5 in the sequence listing; The outer primer pair includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer can be sequence 2 in the sequence listing, and the nucleotide sequence of the downstream primer can be sequence 3 in the sequence listing; the circular primer pair includes upstream A primer and a downstream primer, the nucleotide sequence of the upstream primer can be sequence 6 in the sequence listing, and the nucleotide sequence of the downstream primer can be sequence 7 in the sequence listing.

The Mycoplasma hyopneumoniae detection kit provided by the invention is a kit containing the special primers.

During the use of the Mycoplasma hyopneumoniae detection kit, the inner primer pair, outer primer pair and circular primer pair are used as a whole group, and the formation of primer dimers should be avoided as much as possible during use.

The inner primer pair includes an upstream primer (FIP) and a downstream primer (BIP), the nucleotide sequence of the upstream primer is sequence 4 in the sequence listing, and the nucleotide sequence of the downstream primer is sequence 5 in the sequence listing; the The outer primer pair comprises an upstream primer (F3) and a downstream primer (B3), the nucleotide sequence of the upstream primer is sequence 2 in the sequence listing, and the nucleotide sequence of the downstream primer is sequence 3 in the sequence listing; the circular primer The pair includes an upstream primer (LF) and a downstream primer (LB), the nucleotide sequence of the upstream primer is sequence 6 in the sequence listing, and the nucleotide sequence of the downstream primer is sequence 7 in the sequence listing.

The kit may also include loop-mediated isothermal amplification reagents and sample tubes.

The sample tube is a centrifuge tube containing an FTA card.

The kit can include positive and negative controls.

The positive control can be the sample tube that has adhered the following nucleic acid sequence or adhered to the plasmid containing the following nucleic acid sequence on the FTA card:

Mycoplasma hyopneumoniae DNA, Mycoplasma hyopneumoniae GenBank Accession Number NC-006360 from nucleotides 876699 to 876919 at the 5′ end (partial sequence of 16S ribosomal RNA gene).

The loop-mediated isothermal amplification reagent can specifically be an aqueous solution containing the following solutes: Tris-HCl, KCl, (NH ₄ ) ₂ SO ₄ , Tween20, MgSO ₄ , betaine, calcein, MnCl ₂ , deoxyribonucleotides , Bst DNA polymerase.

Add the inner primer pair, the outer primer pair, and the circular primer pair to the loop-mediated isothermal amplification reagent to form a loop-mediated isothermal amplification reaction solution; each 23 μL of the loop-mediated isothermal amplification In the reaction solution, the amount of the substance of the solute is as follows:

Tris-HCl 0.5μmol, KCl 0.25μmol, (NH ₄ ) ₂ SO ₄ 0.25μmol, Tween20 0.025μL, MgSO ₄ 0.2-20μmol, betaine 20μmol, calcein 0.00125μmol, MnCl ₂ 0.015μmol, 4 kinds of deoxynucleotides Each 0.035 μmol, Bst DNA polymerase 8U, each 0.04-0.06 μmol of the upstream primer and the downstream primer of the inner primer pair, each 0.004-0.008 μmol of the upstream primer and the downstream primer of the outer primer pair, the circular primer pair 0.02-0.04 μmol each of upstream primer and downstream primer;

In every 23 μL of the ring-mediated isothermal amplification reaction solution, the amount of the solute is preferably as follows:

Tris-HCl 0.5 μmol, KCl 0.25 μmol, (NH ₄ ) ₂ SO ₄ 0.25 μmol, Tween20 0.025 μL, MgSO ₄ 0.2 μmol, betaine 20 μmol, calcein 0.00125 μmol, MnCl ₂ 0.015 μmol, each of the four deoxynucleotides 0.035 μmol, Bst DNA polymerase 8U, 0.04 μmol each of the upstream primer and downstream primer of the inner primer pair, 0.004 μmol each of the upstream primer and downstream primer of the outer primer pair, and 0.004 μmol of the upstream primer and downstream primer of the circular primer pair 0.02 μmol each.

The present invention also provides a method for detecting whether Mycoplasma hyopneumoniae is carried in a dead animal, which is to use the special primer or the Mycoplasma hyopneumoniae detection kit to carry out loop-mediated isothermal The amplification reaction is to detect the amplification product to determine whether the dead animal carries Mycoplasma hyopneumoniae; the conditions of the loop-mediated isothermal amplification reaction are: 60-65° C., 20-90 minutes.

The method for detecting whether a dead animal carries Mycoplasma hyopneumoniae further includes the step of reacting the sample after the loop-mediated isothermal amplification reaction at 80-90° C. for 2-10 minutes.

By using the kit provided by the invention to detect Mycoplasma hyopneumoniae, it is possible to determine whether a sample contains Mycoplasma hyopneumoniae by direct inspection. The direct viewing method is:

The reaction tube containing the positive control has bright green visible fluorescence, and the reaction tube containing the negative control is still a brown-yellow non-fluorescent liquid. If the reaction tube containing the sample to be tested is still a brown-yellow non-fluorescent liquid, it means that the test result of Mycoplasma hyopneumoniae in the sample to be tested is negative. If the reaction tube containing the sample to be tested has bright green visible fluorescence, it means that the test result of Mycoplasma hyopneumoniae in the sample is positive.

The Mycoplasma hyopneumoniae may specifically be Mycoplasma hyopneumoniae 232 strain.

The application of the special primers in the preparation of the Mycoplasma hyopneumoniae detection kit also belongs to the protection scope of the present invention.

The detection principle of the mycoplasma hyopneumoniae detection kit provided by the invention is as follows:

According to the Mycoplasma hyopneumoniae genome GenBank Accession Number NC-006360 from the 5' end nucleotides 876699 to 876919 (partial sequence of 16S ribosomal RNA gene) (sequence 1 in the sequence listing) a set of loop-mediated isothermal amplification was designed. Added primers, namely inner primer pair (inner upstream primer FIP and inner downstream primer BIP), outer primer pair (outer upstream primer F3 and outer downstream primer B3) and circular primer pair (circular upstream primer LF and circular downstream primer LB). The three pairs of primers are completely matched with the sequences of the eight specific regions in the partial sequence variation region of Mycoplasma hyopneumoniae, which ensures the complete reaction and the specificity of the method for detecting Mycoplasma hyopneumoniae.

F3: SEQ ID NO 2: AACATCTCACGACACGAG (sequence 2 of the sequence listing);

B3: SEQ ID NO 3: GCTAACGCGTTAAATGATCC (sequence 3 of the sequence listing);

FIP: SEQ ID NO 4: CTTACCCACTCTTGACATTCTCGTTTTCGACAACCATGCACCATC (sequence 4 of the sequence listing);

BIP: SEQ ID NO 5: TAAACCACATGCTCCACCGCTTTTGCCTGAGTAGTATGCTCG (sequence 5 of the sequence listing);

LF: SEQ ID NO 6: AGAGATATAGCCGAGGCTAACGAG (sequence 6 of the sequence listing);

LB: SEQ ID NO 7: TGTGCGGGTTCCCGTCA (sequence 7 of the sequence listing).

The primer set binds to 8 specific regions on the 5' end from 876699 to 876919 nucleotides (16S ribosomal RNA gene partial sequence) in the Mycoplasma hyopneumoniae genome GenBank Accession NumberNC-006360, see Table 1.

Table 1 The binding region of the primer set and the partial sequence of the 16S ribosomal RNA gene

The above primer set can complete the amplification of template DNA under the action of DNA polymerase with high strand displacement catalytic activity. The amplification process is divided into three stages, as follows:

(1) The initial stage of the cycle

One end of the inner primer first binds to the template and initiates DNA synthesis. The outer primer at the same end then binds to the template to initiate DNA synthesis, and strand displacement occurs to release a single strand of DNA containing the sequence of the inner primer. The single-stranded DNA is used as a template to successively combine with the inner primer and the outer primer at the other end to initiate DNA synthesis and strand displacement, and finally form an initial stem-loop DNA.

(2) Reaction cycle stage

The inner primer binds to the initial stem-loop DNA and initiates strand-displacement DNA synthesis, which produces another initial stem-loop DNA and a new double-length stem-loop DNA. These stem-loop DNAs can be used as templates to continue to combine with the inner primers to initiate strand-displacement DNA synthesis, and each synthesis can double the stem length of the stem-loop DNA.

After entering the cycle stage, many stem-loop DNAs with different molecular sizes will be produced, and these stem-loop DNAs can also be used as templates to bind with circular primers to initiate more DNA synthesis and strand displacement. Finally, after an hour of isothermal amplification, the target DNA can accumulate 10 ⁹ copies.

The Mycoplasma hyopneumoniae detection kit provided by the invention is used for detection, which has high specificity and high sensitivity, and can detect 10 copies of the target DNA. Compared with the PCR detection method, the application of the Mycoplasma hyopneumoniae detection kit provided by the present invention does not require an expensive PCR instrument, but only an ordinary metal or water bath, and the detection result can be detected by observing the visible fluorescence, and the operation is simple convenient. The kit of the present invention can be applied to the clinical medical detection at the grassroots level and the detection of mycoplasma hyopneumoniae that may be contaminated in food, and is especially suitable for the clinical medical detection at the grassroots level and on-site real-time detection.

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The kit provided by the invention detects Mycoplasma hyopneumoniae and can comprise the following steps:

(1) Processing of samples

① Sample to be tested

Put the collected nasal swab into a 1.5ml centrifuge tube, add 1ml of normal saline to the tube, vortex for 30s, take out the nasal swab; centrifuge the remaining liquid at 10000rpm for 1 minute; discard the supernatant, leaving only 100μl of liquid, Shake until the bacteria are completely suspended to obtain a liquid sample. Take 10 μl of liquid sample and drop it on the FTA card of the sample tube, incubate for 10 minutes, suck out the liquid with the pipette, leave a piece of paper, add 200 μl of ddH ₂ O, and incubate at room temperature for 5 minutes (if necessary, appropriate manual mixing), use a pipette to suck out all the used ddH ₂ O in the tube to obtain.

② Negative control

Add 10 μl of double-distilled water dropwise to the FTA card in the sample tube, and after standing at room temperature for 10 minutes, use a pipette to suck out the liquid, leaving a piece of paper. Add 200 μl ddH ₂ O and let stand at room temperature for 5 minutes. Use a pipette to suck out all the used ddH ₂ O in the tube, and the product is obtained.

(2) Loop-mediated isothermal amplification

① Add 23 μl of LAMP reaction solution to the sample tube (the 2mm FTA card is regarded as 2 μl in the reaction system);

②Place it in a water bath or a metal constant temperature heater at 60-65°C for 20-90 minutes, then take it out.

After the completion of step ②, before taking it out, you can also adjust the temperature of the water bath to 80-90°C for 2-10 minutes to react for 2-10 minutes. The purpose is to terminate the reaction and store the reaction results for a long time.

(3) Result judgment

The presence or absence of M. hyopneumoniae in a sample can be determined by direct inspection.

The reaction tube containing the positive control has bright green visible fluorescence, and the reaction tube containing the negative control is still a brown-yellow non-fluorescent liquid. If the reaction tube containing the sample to be tested is still a brown-yellow non-fluorescent liquid, it means that the test result of Mycoplasma hyopneumoniae in the sample to be tested is negative. If the reaction tube containing the sample to be tested has bright green visible fluorescence, it means that the test result of Mycoplasma hyopneumoniae in the sample is positive.

Embodiment 1, the preparation of Mycoplasma hyopneumoniae detection kit

1. Synthesis of primers

Artificially synthesize the following 3 pairs of primers:

F3: AACATCTCACGACACGAG;

B3: GCTAACGCGTTAAATGATCC;

FIP: CTTACCCACTCTTGACATTCTCGTTTTCGACAACCATGCACCATC;

BIP: TAAACCACATGCTCCACCGCTTTTGCCTGAGTAGTATGCTCG;

LF:AGAGATATAGCCGAGGCTAACGAG;

LB: TGTGCGGGTTCCCGTCA.

2. Preparation of sample tube

Use a 2.0mm perforated sampler to remove small discs with a diameter of 2.0mm from a blank FTA card (Whatman, Cat No.WB120205), and place them at the bottom of a 0.25ml centrifuge tube.

3. Preparation of positive control

The 232 strains of Mycoplasma hyopneumoniae (China Veterinary Drug Administration) were diluted with normal saline and inoculated into healthy pigs. Two weeks later, the diseased lung was collected with aseptic technique, and the pathogen was isolated and cultured by the "disease lung block soaking method" and examined under a microscope. Typical bacteria of Mycoplasma hyopneumoniae were observed. Add Hank's solution (20ml Hank's solution per 1g of lung tissue) to the diseased lung, mash it to make a homogenate, add penicillin G and thallium acetate, and centrifuge to make a sterile diseased lung suspension. Take 10 μl of aseptic lung suspension, drop it on a 2.0mm FTA card, incubate for 10 minutes, suck out the bacterial solution with a pipette, leave a piece of paper, add 200 μl ddH ₂ O, and incubate at room temperature for 5 minutes (if necessary, Proper manual mixing can be carried out in the tube), and all the used ddH ₂ O in the tube is sucked out with a pipette, and the mycoplasma DNA adheres to the treated FTA card. The positive control was obtained and stored at -20 to -4°C for future use.

4. Preparation of LAMP reaction solution

Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH ₄ ) ₂ SO ₄ , 0.025 μL Tween20, 0.2 μmol MgSO ₄ , 20 μmol Betaine, 0.00125 μmol calcium Chlorophyll, 0.015 μmol MnCl ₂ , four deoxynucleotides (dNTPs) each 0.035 μmol, 0.04 μmol upstream inner primer (FIP), 0.04 μmol downstream inner primer (BIP), 0.004 μmol upstream outer primer (F3), 0.004 μmol downstream Outer primer (B3), 0.02 μmol upstream circular primer (LF), 0.02 μmol downstream circular primer (LB), 8 U of Bst DNA polymerase, sterile double distilled water.

5. Assembling the kit

The kit consists of the following materials: the LAMP reaction solution prepared in step 4, the positive control prepared in step 3 and several sample tubes prepared in step 2.

Embodiment 2, the preparation of Mycoplasma hyopneumoniae detection kit

1. Synthesis of primers

Same as Step 1 of Example 1.

2. Preparation of sample tube

Same as Step 2 of Example 1.

3. Preparation of positive control

Same as Step 3 of Example 1.

4. Preparation of LAMP reaction solution

Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH ₄ ) ₂ SO ₄ , 0.025 μL Tween20, 20 μmol MgSO ₄ , 20 μmol Betaine, 0.00125 μmol calcein , 0.015 μmol MnCl ₂ , 0.035 μmol each of the four deoxynucleotides (dNTPs), 0.06 μmol upstream inner primer (FIP), 0.06 μmol downstream inner primer (BIP), 0.008 μmol upstream outer primer (F3), 0.008 μmol downstream outer primer Primer (B3), 0.04 μmol upstream circular primer (LF), 0.04 μmol downstream circular primer (LB), 8 U of Bst DNA polymerase, sterile double distilled water.

5. Assembling the kit

Same as Step 5 of Example 1.

Embodiment 3, the preparation of Mycoplasma hyopneumoniae detection kit

1. Synthesis of primers

Same as Step 1 of Example 1.

2. Preparation of sample tube

Same as Step 2 of Example 1.

3. Preparation of positive control

Same as Step 3 of Example 1.

4. Preparation of LAMP reaction solution

Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH ₄ ) ₂ SO ₄ , 0.025 μL Tween20, 10 μmol MgSO ₄ , 20 μmol Betaine, 0.00125 μmol calcein , 0.015 μmol MnCl ₂ , 0.035 μmol each of the four deoxynucleotides (dNTPs), 0.05 μmol upstream inner primer (FIP), 0.05 μmol downstream inner primer (BIP), 0.006 μmol upstream outer primer (F3), 0.006 μmol downstream outer primer Primer (B3), 0.03 μmol upstream circular primer (LF), 0.03 μmol downstream circular primer (LB), 8 U of Bst DNA polymerase, sterile double distilled water.

5. Assembling the kit

Same as Step 3 of Example 1.

Embodiment 4, the application of Mycoplasma hyopneumoniae detection kit

Two pigs were taken, one pig confirmed to be infected with Mycoplasma hyopneumoniae and one healthy pig. The nasal secretion swabs were collected from two pigs respectively, and the samples were detected by using the kit prepared in Example 1.

1. Sample processing

1. Sample to be tested

Put the collected nasal swab into a 1.5ml centrifuge tube, add 1ml of normal saline to the tube, vortex for 30s, take out the nasal swab; centrifuge the remaining liquid at 10000rpm for 1 minute; discard the supernatant, leaving only 100μl of liquid, Shake until the bacteria are completely suspended to obtain a liquid sample. Take 10 μl of liquid sample and drop it on the FTA card of the sample tube, incubate for 10 minutes, suck out the liquid with the pipette, leave a piece of paper, add 200 μl of ddH ₂ O, and incubate at room temperature for 5 minutes (if necessary, appropriate manual mixing), use a pipette to suck out all the used ddH ₂ O in the tube to obtain.

The swabs of nasal secretions of sick pigs were used for the above treatment, and three sample tubes were obtained, which were used as group A sample tubes.

The nasal secretion swabs of healthy pigs were used for the above treatment to obtain three sample tubes, which were used as group B sample tubes.

2. Negative control

Add 10 μl of double-distilled water dropwise to the FTA card in the sample tube, and after standing at room temperature for 10 minutes, use a pipette to suck out the liquid, leaving a piece of paper. Add 200 μl ddH ₂ O and let stand at room temperature for 5 minutes. Use a pipette to suck out all the used ddH ₂ O in the tube, and the product is obtained.

2. Loop-mediated isothermal amplification

In group A sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 1; in group B sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 2; take positive 3 tubes each of control and negative control were added with 23 μL of LAMP reaction solution.

The above-mentioned sampling tubes were placed on the water bath at 60°C for 90 minutes at the same time, and then taken out.

3. View

The sample tube containing the positive control has bright green visible fluorescence, and the sample tube containing the negative control is still a brown-yellow non-fluorescent liquid. The three reaction tubes in the healthy pig test group were all brownish yellow, and the three reaction tubes in the sick pig test group had bright green visible fluorescence.

Embodiment 5, application of Mycoplasma hyopneumoniae detection kit

Two pigs were taken, one pig confirmed to be infected with Mycoplasma hyopneumoniae and one healthy pig. The nasal secretion swabs were collected from two pigs respectively, and the samples were detected by using the kit prepared in Example 3.

1. Sample processing

Same as Step 1 of Example 4.

2. Loop-mediated isothermal amplification

In group A sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 1; in group B sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 2; take positive 3 tubes each of control and negative control were added with 23 μL of LAMP reaction solution.

The above-mentioned sampling tubes were placed on the water bath at 65°C for 70 minutes at the same time, and then taken out.

3. View

The sample tube containing the positive control has bright green visible fluorescence, and the sample tube containing the negative control is still a brown-yellow non-fluorescent liquid. The three reaction tubes in the healthy pig test group were all brownish yellow, and the three reaction tubes in the sick pig test group had bright green visible fluorescence.

Embodiment 6, application of Mycoplasma hyopneumoniae detection kit

Two pigs were taken, one pig confirmed to be infected with Mycoplasma hyopneumoniae and one healthy pig. The nasal secretion swabs were collected from two pigs respectively, and the samples were detected by using the kit prepared in Example 2.

1. Sample processing

Same as Step 1 of Example 4.

2. Loop-mediated isothermal amplification

In group A sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 1; in group B sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 2; take positive 3 tubes each of control and negative control were added with 23 μL of LAMP reaction solution.

The above-mentioned sampling tubes were placed on the water bath at 60°C for 50 minutes at the same time, and then taken out.

3. View

The sample tube containing the positive control has bright green visible fluorescence, and the sample tube containing the negative control is still a brown-yellow non-fluorescent liquid. The three reaction tubes in the healthy pig test group were all brownish yellow, and the three reaction tubes in the sick pig test group had bright green visible fluorescence.

Embodiment 7, application of Mycoplasma hyopneumoniae detection kit

Two pigs were taken, one dead pig confirmed to be infected with Mycoplasma hyopneumoniae, and one healthy pig. The nasal secretion swabs were collected from two pigs respectively, and the samples were detected by using the kit prepared in Example 1.

1. Sample processing

Same as Step 1 of Example 4.

2. Loop-mediated isothermal amplification

In group A sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 1; in group B sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 2; take positive 3 tubes each of control and negative control were added with 23 μL of LAMP reaction solution.

At the same time, the above-mentioned sampling tubes were placed on the water bath at 65°C for 30 minutes, and the temperature of the water bath was adjusted to 80°C for 10 minutes, and then taken out.

3. View

The sample tube containing the positive control has bright green visible fluorescence, and the sample tube containing the negative control is still a brown-yellow non-fluorescent liquid. The three reaction tubes in the healthy pig detection group were all brownish yellow, and the three reaction tubes in the dead pig detection group all had bright green visible fluorescence.

Embodiment 8, application of Mycoplasma hyopneumoniae detection kit

Two pigs were taken, one dead pig confirmed to be infected with Mycoplasma hyopneumoniae, and one healthy pig. The nasal secretion swabs were collected from two pigs respectively, and the samples were detected by using the kit prepared in Example 2.

1. Sample processing

Same as Step 1 of Example 4.

2. Loop-mediated isothermal amplification

In group A sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 1; in group B sample tubes, add 23 μL LAMP reaction solution to each tube, as test group 2; take positive 3 tubes each of control and negative control were added with 23 μL of LAMP reaction solution.

At the same time, place the above-mentioned sampling tube on the water bath at 60°C for 20 minutes, adjust the temperature of the water bath to 90°C and react for 2 minutes, then take it out.

3. View

The sample tube containing the positive control has bright green visible fluorescence, and the sample tube containing the negative control is still a brown-yellow non-fluorescent liquid. The three reaction tubes in the healthy pig detection group were all brownish yellow, and the three reaction tubes in the dead pig detection group all had bright green visible fluorescence.

Embodiment 9, the detection of the sensitivity of Mycoplasma hyopneumoniae detection kit

Prepare 0.5×10 ⁴ copies/μL, 0.5×10 ³ copies/μL, 0.5×10 ² copies/μL, 5 copies/μL and 0.5 copies/μL of plasmid samples carrying the partial sequence of the 16S ribosomal RNA gene of Mycoplasma hyopneumoniae. The plasmid sample was detected using the kit prepared in Example 3.

1. Preparation of plasmids

The total DNA of 232 strains of Mycoplasma hyopneumoniae (China Veterinary Drug Administration) was extracted, and the partial sequence of 16S ribosomal RNA gene was amplified according to the PCR method (see sequence 1 in the sequence list). After being connected with the T carrier, a plasmid carrying a partial sequence of the 16S ribosomal RNA gene is formed, and one plasmid molecule corresponds to one copy of the partial sequence of the 16S ribosomal RNA gene. The plasmid is transferred into the competent cells, and the competent cells are cultivated under suitable conditions, and the plasmid can be replicated along with the reproduction of the competent cells. Finally, the purified plasmid was extracted from competent cells. The resulting plasmid can be measured spectrophotometrically and the copy number concentration calculated. Dilute the plasmid to the required concentration with double distilled water, namely 0.5×10 ⁴ copies/μL, 0.5× ^{10 3} copies/μL, 0.5× ^{10 2} copies/μL, 5 copies/μL and 0.5 copies/μL. Add 2 μL of each concentration of plasmid during detection, and each reaction contains 10 ⁴ copies/μL, 10 ³ copies/μL, 10 ² copies/μL, 10 copies/μL, and 1 copy/μL of plasmid samples. (Refer to the third edition of "Guidelines for Molecular Cloning Experiments", and the required reagents and instruments are commercially available.)

2. Detection of Mycoplasma hyopneumoniae

1. Preparation of negative control

For the negative control, 10 μl of double-distilled water was added dropwise on the FTA card in the sample tube, and after standing at room temperature for 10 minutes, the liquid was sucked out with a pipette, leaving a piece of paper. Add 200 μl ddH ₂ O and let stand at room temperature for 5 minutes. Use a pipette to suck out all the used ddH ₂ O in the tube, and the product is obtained.

2. Loop-mediated isothermal amplification

Take 0.5×10 ⁴ , 0.5×10 ³ , 0.5×10 ² copies/μL, 5 copies/μL and 0.5 copies/μL copies/μL of the plasmid samples obtained in step 1. Add 2 μL each to 3 sample tubes, Add 23 μL of LAMP reaction solution to each tube as test groups 1, 2, 3, 4 and 5 respectively; take 3 tubes of positive control and negative control, and add 23 μL of LAMP reaction solution respectively.

At the same time, the above-mentioned sampling tubes were placed on the water bath at 65°C for 50 minutes, and the temperature of the water bath was adjusted to 80°C for 10 minutes, and then taken out.

3. Direct view

The results showed that the three reaction tubes in the positive control group all had bright green visible fluorescence, and the three reaction tubes in the negative control group were all brown-yellow non-fluorescent liquid. The three reaction tubes of detection group 1 all have bright green visible fluorescence, the three reaction tubes of detection group 2 all have bright green visible fluorescence, the three reaction tubes of detection group 3 all have bright green visible fluorescence, and the three reaction tubes of detection group 4 have bright green visible fluorescence. Each of the reaction tubes had bright green visible fluorescence, and the three reaction tubes of test group 5 were all brown-yellow non-fluorescent liquids.

sequence listing

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<210>5

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>5

<210>6

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>6

<210>7

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>7

Claims (10)

1. The special primers for detecting Mycoplasma hyopneumoniae consist of the following three pairs of primers for loop-mediated isothermal amplification: one pair of primers is the inner primer pair combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360, one A pair of primers is a pair of outer primers combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360, and a pair of primers is a pair of circular primers combined with the 16S ribosomal RNA gene in Mycoplasma hyopneumoniae GenBank Accession Number NC-006360. 2. The special primer according to claim 1, characterized in that: among the special primers, a pair of primers can be combined with the 5′ end from the 876699th to 876919th core of the Mycoplasma hyopneumoniae genome GenBank Accession Number NC-006360. A pair of inner primers in the nucleotide region, a pair of primers is a pair of outer primers that can bind to the nucleotide region from 876699 to 876919 at the 5′ end in GenBank Accession Number NC-006360 of the Mycoplasma hyopneumoniae genome, and a pair of primers can bind to A pair of circular primers in the nucleotide region from 876699 to 876919 at the 5′ end in GenBank AccessionNumber NC-006360 of the Mycoplasma hyopneumoniae genome. 3. The special primer according to claim 2, characterized in that: said inner primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is sequence 4 in the sequence table, and the nucleotide sequence of the downstream primer is is sequence 5 in the sequence listing; the outer primer pair includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is sequence 2 in the sequence listing, and the nucleotide sequence of the downstream primer is sequence 3 in the sequence listing; The circular primer pair includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is sequence 6 in the sequence listing, and the nucleotide sequence of the downstream primer is sequence 7 in the sequence listing. 4. A detection kit for Mycoplasma hyopneumoniae containing the special primer according to any one of claims 1 to 3. 5. The kit according to claim 4, characterized in that: the kit further comprises a loop-mediated isothermal amplification reagent and a sample tube; the sample tube is a centrifuge tube containing an FTA card. 6. The kit according to claim 5, characterized in that: said loop-mediated isothermal amplification reagent is an aqueous solution containing the following solutes: Tris-HCl, KCl, (NH ₄ ) ₂ SO ₄ , Tween20, MgSO ₄ , betaine, calcein, MnCl ₂ , deoxyribonucleotides, Bst DNA polymerase. 7. The kit according to claim 6, wherein the inner primer pair, the outer primer pair and the circular primer pair are added to the loop-mediated isothermal amplification reagent to form a loop-mediated amplification reagent. Isothermal amplification reaction solution; each 23 μL of the ring-mediated isothermal amplification reaction solution contains the following solutes: Tris-HCl 0.5μmol, KCl 0.25μmol, (NH ₄ ) ₂ SO ₄ 0.25μmol, Tween20 0.025μL, MgSO ₄ 0.2-20μmol, betaine 20μmol, calcein 0.00125μmol, MnCl ₂ 0.015μmol, 4 kinds of deoxynucleotides Each 0.035 μmol, Bst DNA polymerase 8U, each 0.04-0.06 μmol of the upstream primer and the downstream primer of the inner primer pair, each 0.004-0.008 μmol of the upstream primer and the downstream primer of the outer primer pair, the circular primer pair 0.02-0.04 μmol each of upstream primer and downstream primer; In every 23 μL of the ring-mediated isothermal amplification reaction solution, the MgSO is preferably _0.2 μmol, the upstream primer and downstream primer of the inner primer pair are preferably 0.04 μmol, and the upstream primer and downstream primer of the outer primer pair are preferably 0.004 μmol. μmol, the upstream primer and the downstream primer of the circular primer pair are preferably 0.02 μmol. 8. A method for detecting whether Mycoplasma hyopneumoniae is carried in a dead animal, comprising the steps of: using the special primer according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 7 on the dead animal The total DNA of the animal is subjected to a loop-mediated isothermal amplification reaction, and the amplified product is detected to determine whether the dead animal carries Mycoplasma hyopneumoniae; the conditions of the loop-mediated isothermal amplification reaction are: 60-65°C, 20-90 minutes . 9. The method according to claim 8, characterized in that: the method for detecting whether dead animals carry Mycoplasma hyopneumoniae also includes reacting the sample that has undergone the loop-mediated isothermal amplification reaction at 80-90°C 2-10 minute steps. 10. The application of the special primer according to any one of claims 1 to 3 in the preparation of a detection kit for Mycoplasma hyopneumoniae.