Sheep mycoplasmal pneumonia pathogen detection kit and application thereof

Abstract

The invention relates to the field of pathogen detection, and particularly discloses a sheep mycoplasmal pneumonia pathogen detection kit and application thereof. By designing a specific primer with high sensitivity and using a special DNA extraction reagent, high-quality mycoplasma DNA can be efficiently purified, a unique gene segment of PCR amplification targeted sheep mycoplasma is ensured, the problem of non-specific amplification or primer failure is effectively avoided, and the reliability of a detection result is ensured; the optimized cracking and purifying process can recover pathogen DNA from a small amount of disease materials to the maximum extent, pathogen loading capacity in a sample is low, the pathogen can be effectively detected, and the occurrence probability of false positive or false negative can be effectively reduced; meanwhile, the kit greatly shortens the detection time, is used for rapid diagnosis of a large number of clinical samples infected by mycoplasma pneumonia of sheep, and improves the detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of pathogen detection, specifically relating to a kit for detecting Mycoplasma pneumoniae pathogens in sheep and its application. Background Technology

[0002] Mycoplasma pneumoniae in sheep is a highly contagious respiratory disease caused by Mycoplasma sheepii. It is primarily transmitted through airborne droplets from coughs and sneezes of infected sheep, as well as through close contact with infected animals. Goats of all ages are susceptible, especially lambs and young sheep, although sheep can also be infected. Common symptoms in affected sheep include fever, loss of appetite, coughing, and nasal discharge. Severe cases involve rapid and difficult breathing, resembling a dog-sitting posture, groaning, and serous-bloody nasal discharge. Death usually occurs within 12-36 hours due to suffocation. Post-mortem examination of infected sheep reveals large amounts of pale yellow, turbid pleural effusion; the pleura becomes rough and thickened, adhering to the pleura and pericardium; the lungs are edematous and distended, showing hepatic areas of different colors (red, gray, yellow) with a marbled appearance; necrotic foci of varying sizes are present in the lung lobes, surrounded by connective tissue, exhibiting a layered structure on cut surface.

[0003] Currently, a preliminary diagnosis can be made based on typical epidemiological symptoms (high fever, cough, rusty nasal discharge, and difficulty breathing) and characteristic pathological changes (marbled lungs and pleural effusion). However, by the time these symptoms appear, the disease has generally spread extensively, posing a challenge to treatment. Conventional biochemical and serological testing methods are prone to false positives or false negatives, affecting the accuracy of the results. Therefore, establishing a highly efficient, sensitive, specific, and rapid method for detecting ovine mycoplasma pneumoniae is of paramount importance. Summary of the Invention

[0004] To address the shortcomings and problems existing in the current detection of Mycoplasma pneumoniae pathogens in sheep, this invention provides a Mycoplasma pneumoniae pathogen detection kit and its application.

[0005] This invention provides a method for detecting the pathogen of ovine mycoplasma pneumonia, characterized by comprising the following steps: S1. Sample collection and DNA extraction; S2. Specific primer design, wherein the primers are: MccpF: 5'-CTTCAAGATCATTATTAATCC-3'; MccpR: 5'-AATATATGCATTACTATGAGTAATTAT-3'; S3, PCR amplification and detection of amplification products.

[0006] The above-mentioned method for detecting the pathogen of ovine mycoplasma pneumoniae consists of the following PCR amplification system: DNA 5 μl, MccpF 2 μl, MccpR 2 μl, 5×PCR Buffer 5 μl, 2.5 mmol / L dNTPs Mix 5 μl, Taq enzyme 0.5 μl, 25 mg / L MgCl2 3.5 μl, and sterile ultrapure water 2 μl.

[0007] The above-mentioned method for detecting the pathogen of ovine mycoplasma pneumoniae uses a PCR amplification program of: 95℃ for 30s; then 30 cycles: 94℃ for 5s, 55℃ for 5s, 72℃ for 5s; and finally 72℃ for 5min.

[0008] The above-mentioned method for detecting the pathogen of ovine mycoplasma pneumonia uses a PCR amplification length of 310 bp.

[0009] The present invention also provides a kit for detecting Mycoplasma pneumoniae pathogen in sheep, comprising lysis buffer, washing buffer 1, washing buffer 2, sterile double-purified water, dNTPs, MgCl2, reaction buffer 5×PCR Buffer, upstream primer, downstream primer, Taqenzyme, positive control and negative control.

[0010] The above-mentioned detection kit for mycoplasma pneumoniae pathogens in sheep has been applied to the detection of mycoplasma pneumoniae pathogens.

[0011] Compared with existing technologies, the beneficial effects of this invention are as follows: By designing highly sensitive specific primers and using dedicated DNA extraction reagents, this invention can efficiently purify high-quality mycoplasma DNA, ensuring that PCR amplification of unique gene fragments targeting Mycoplasma sheep effectively avoids the problems of non-specific amplification or primer failure, thus guaranteeing the reliability of detection results; the optimized lysis and purification process of this invention can maximize the recovery of pathogen DNA from small amounts of pathogenic material, and even if the pathogen load in the sample is low, it can still be effectively detected, which can effectively reduce the probability of false positives or false negatives; at the same time, the kit of this invention greatly shortens the detection time, making it suitable for the rapid diagnosis of large numbers of clinical samples infected with Mycoplasma sheep pneumonia pathogens, thus improving detection efficiency. Attached Figure Description

[0012] Figure 1 This is a diagram showing the PCR amplification results of this invention. Detailed Implementation

[0013] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0014] Example 1: This example provides a method for detecting the pathogen of ovine mycoplasma pneumonia, including the following steps: (1) Primer design: A pair of specific primers were designed and screened using Mccp-specific sequences as target genes. MccpF: 5'-CTTCAAGATCATTATTAATCC-3'; MccpR: 5'-AATATATGCATTACTATGAGTAATTAT-3'.

[0015] (2) Sample collection: ① Pathological samples: Collect 0.1 mg to 100 mg of pathological samples from sheep aseptically and put them into sterile centrifuge tubes for later use.

[0016] ② Nasopharyngeal swab: Collect nasopharyngeal fluid using a nasopharyngeal swab and place it in a sterile centrifuge tube for later use.

[0017] ③ Blood: Take 10 μl to 100 μl of whole blood, serum or plasma and put it into a sterile centrifuge tube for later use.

[0018] (3) Extraction of DNA from the sample ① Solid animal tissues are first ground up, while liquid animal tissues are used directly for extraction; ② Take 10 μl to 100 μl of liquid or 0.1 mg to 100 mg of ground solid animal tissue, add 400 to 600 μl of DNA lysis buffer, and separate the supernatant at 12000 rpm for 30 s. ③ Transfer the supernatant to a centrifuge tube, add 500 μl of buffer, transfer it to the DNA purification column in portions, and centrifuge at 12000 rpm for 30 s; discard the waste liquid in the collection tube. ④ Rinse the purification column with 400~600 μl of washing solution 1, centrifuge at 12000 rpm for 30 s, and discard the waste liquid in the collection tube; ⑤ Rinse the purification column with 500~700 μl of washing solution 2, centrifuge at 12000 rpm for 30 s, and discard the waste liquid in the collection tube; ⑥ Transfer the DNA purification column to another sterile centrifuge tube, add 50 μl of sterile ultrapure water, centrifuge at 12000 for 30 s to obtain high-purity DNA, and store at -20 ℃ for later use.

[0019] (4) PCR amplification The reaction system consisted of 25 μl of DNA. 5 μl of extracted DNA was added to 2 μl of MccpF (25 pmol / μl), 2 μl of MccpR (25 pmol / μl), 5 μl of 5×PCR Buffer, 5 μl of 2.5 mmol / L dNTPs, 0.5 μl of Taq enzyme (5 U / μl), and 1.5 μl of 25 mM MgCl2. 1 μl of positive control, 1 μl of negative control, and 2 μl of sterile ultrapure water were mixed thoroughly and then briefly centrifuged.

[0020] The PCR amplification procedure is as follows:

[0021] (5) Detection of PCR products Mix 5 μl of amplification product with 2 μl of loading buffer, add to a 1.0% agarose gel containing EB, and simultaneously add the control and DNA marker (100 bp). Electrophoresis is performed at 120 V for 35 min, and the results are then observed using a gel imaging system. Figure 1 As shown, the amplified product length is 310 bp.

[0022] Example 2: This example provides a kit for detecting the pathogen of ovine mycoplasma pneumoniae. The kit includes, DNA extraction reagents include lysis buffer, wash buffer 1, wash buffer 2 and elution buffer, wherein the lysis buffer includes 5~15 mM EDTA, 2~5% (by weight) polyvinylpyrrolidone, 0.5~1.5% (by weight) hexadecyltrimethylamine bromide and 40~60 mM Tris-HCl. Rinse solution 1: 7~14 mM EDTA, 75% (v / v) ethanol, pH 7.0; Rinse solution 2: 30~50 mM Tris-HCl, 80% (v / v) ethanol, pH 7.0.

[0023] PCR amplification reagents: including 5 μl of 5×PCR Buffer, 5 μl of 2.5 mmol / L dNTP Mix, 0.5 μl of Taq enzyme (5 U / μl), 1.5 μl of 25 mM MgCl2, 2 μl of MccpF (25 pmol / μl), 2 μl of MccpR (25 pmol / μl), 1 μl of positive control, 1 μl of negative control, and 2 μl of sterile ultrapure water.

[0024] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting the pathogen of ovine mycoplasmal pneumonia, characterized in that: Includes the following steps: S1. Sample collection and DNA extraction; S2. Specific primer design, wherein the primers are: MccpF: 5'-CTTCAAGATCATTATTAATCC-3'; MccpR: 5'-AATATATGCATTACTATGAGTAATTAT-3'; S3, PCR amplification and detection of amplification products.

2. The method for detecting the pathogen of ovine mycoplasma pneumoniae according to claim 1, characterized in that: The PCR amplification system consisted of: 5 μl DNA, 2 μl MccpF, 2 μl MccpR, 5 μl 5×PCR Buffer, 5 μl 2.5 mmol / L dNTPs Mix, 0.5 μl Taq enzyme, 3.5 μl 25 mM MgCl2, and 2 μl sterile ultrapure water.

3. The method for detecting the pathogen of ovine mycoplasma pneumoniae according to claim 1, characterized in that: The PCR amplification program was as follows: 95℃ for 30 seconds; then 30 cycles: 94℃ for 5 seconds, 55℃ for 5 seconds, 72℃ for 5 seconds; and finally 72℃ for 5 minutes.

4. The method for detecting the pathogen of ovine mycoplasma pneumoniae according to claim 1, characterized in that: The PCR amplification length was 310 bp.

5. A kit for detecting the pathogen of ovine mycoplasma pneumonia, characterized in that: The mixture includes lysis buffer, wash buffer 1, wash buffer 2, sterile double-purified water, dNTPs, MgCl2, 5×PCR Buffer, upstream primer, downstream primer, Taq enzyme, positive control, and negative control.

6. The application of the ovine mycoplasma pneumoniae pathogen detection kit according to claim 5 in the detection of ovine mycoplasma pneumoniae pathogens.

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