Triple PCR kit for diagnosing FHT/MP/HPS and detection method of triple PCR kit

A detection method, FHT technology, applied in the field of molecular biology diagnosis of animal diseases in veterinary medicine, can solve the problems such as the development and report of triple PCR detection technology that have not been seen, and achieve the effects of shortening diagnosis time, saving diagnosis and high sensitivity

Active Publication Date: 2020-05-19
XIANYANG VOCATIONAL TECHN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogens of the above three epidemic diseases are pathogenic microorganisms that are difficult to cultivate or cannot yet be cultivated. Researchers have also established PCR diagnostic methods for related pathogens, but there is no report on the development of triple PCR detection technology. Therefore, a rapid identification of FHT / The diagnostic kit for MP / HPS infection is of great practical significance for rapid clinical diagnosis of corresponding diseases

Method used

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  • Triple PCR kit for diagnosing FHT/MP/HPS and detection method of triple PCR kit
  • Triple PCR kit for diagnosing FHT/MP/HPS and detection method of triple PCR kit
  • Triple PCR kit for diagnosing FHT/MP/HPS and detection method of triple PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] A triple PCR kit for diagnosing FHT / MP / HPS, including lysate, amplification reaction mixture, negative control and positive control, specifically as follows:

[0051] 1) Lysis solution: the composition is DNAiso Reagent, 20 reactions total 20mL, packed into 1 bottle;

[0052] 2) Amplification reaction mixture: composed of sterilized triple distilled water, 10×PCR Buffer, 2.5mmol·L -1 dNTP, 25μmol·L -1 FHT-F upstream primer, 25 μmol L -1 FHT-R downstream primer, 25 μmol L -1 MP-F upstream primer, 25 μmol L -1 MP-R downstream primer, 25 μmol L -1 HPS-F upstream primer, 25 μmol L -1 HPS-R downstream primer, 5U / μL rTaq DNA polymerase, each reaction volume ratio is 15.0︰2.5︰2︰0.5︰0.5︰0.5︰0.5︰0.5︰0.5︰0.5, a total of 23μL, 20 reactions total 460μL, Packed into 1 tube; the design and preparation of the primers are as follows:

[0053] Referring to the genome sequences of Eperythrozoon suis, Mycoplasma suis pneumonia, and Haemophilus parasuis published on GenBank, after c...

Embodiment 2

[0088] 1. Processing of tissue disease samples

[0089] Take 3-5g of tissue disease materials from suspected cases, such as lung, liver or spleen, cut into paste, add 5 times of sterile PBS buffer, grind thoroughly with a tissue grinder, collect the suspension, and centrifuge at 4°C, 12000r / min for 10min , absorb the supernatant, and set aside;

[0090] 2. DNA extraction

[0091] 2.1 DNA extraction from tissue disease samples: draw 100 μL of the obtained supernatant into a 1.5 mL sterile EP tube, add 800 μL of lysate, mix evenly by inversion, let stand for lysis for 10 min, add 600 μL of ice-cold absolute ethanol, mix upside down, Place the pellet for 10 min, centrifuge at 12,000 r / min for 10 min, discard the supernatant, wash once with 75% ethanol, discard the ethanol, invert the EP tube, dry naturally in the air, and use 40 μL of 8 mmol·L -1 NaOH was dissolved to obtain DNA extraction solution of tissue disease material;

[0092] 2.2 Negative control sample extraction: dr...

Embodiment 3

[0105] 1. Processing of serum samples

[0106] Take blood from the anterior vena cava of pigs with suspected cases, and naturally coagulate it. After the serum is separated out, the serum is separated for use;

[0107] 2. DNA extraction, PCR amplification reaction and electrophoresis detection

[0108] The specific detection method is the same as that in Example 2, except that the tissue disease sample in Example 2 is replaced by a serum sample.

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Abstract

The invention belongs to the field of veterinary animal epidemic disease molecular biological diagnosis and discloses a triple PCR kit for diagnosing FHT/MP/HPS and a detection method of the triple PCR kit. The kit comprises 3 pairs of specific primers, wherein FHT-F/FHT-R is an eperythrozoon suis specific amplification primer group, MP-F/MP-R is a mycoplasma hyopneumoniae specific amplification primer group, and HPS-F/HPS-R is a haemophilus parasuis specific amplification primer group; and corresponding specific genome segments can be amplified from epidemic materials of a sick pig, single infection or mixed infection of three pathogens including eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis can be confirmed in once detection. The kit has high detection sensitivity, good specificity, high stability and intuitive result, and the detection method is easy to operate, is convenient and quick, can greatly shorten the detection time and provides technical support toclinical infection control.

Description

technical field [0001] The invention relates to the field of molecular biology diagnosis of animal diseases in veterinary medicine, in particular to a triple PCR kit for diagnosing FHT / MP / HPS and a detection method thereof, which are used for Eperythrozoon porcine, Mycoplasma suis pneumonia, and Haemophilus parasuis detection. Background technique [0002] Eperythrozoon suis can cause fever, anemia and jaundice in pig herds. In recent years, the disease has been commonly infected in pig farms, causing large economic losses to the pig industry. Studies suggest that vertical transmission and hematogenous transmission are the main ways of infection of Eperythrozoon. Eperythrozoon cannot be purely cultured in vitro, so it is difficult to diagnose porcine Eperythrozoon disease. The misdiagnosis rate of current diagnosis by microscopic examination is relatively high. To improve the accuracy and specificity of diagnosis, it is necessary to use molecular biology techniques. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/21C12R1/35C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143Y02A50/30
Inventor 朱小甫吴旭锦
Owner XIANYANG VOCATIONAL TECHN COLLEGE
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