A rapid detection method and primer composition of trichinella spiralis based on lamp technology

A primer composition and technology of Trichinella spiralis, applied in the fields of veterinary biotechnology and molecular biology, can solve the problems of high labor intensity of staff, low infection intensity of meat products, high price of PCR instrument, etc., and achieve simple operation and short time required , the effect of high sensitivity

Active Publication Date: 2018-03-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, microscopic examination and digestion are mainly used in the quarantine of animal trichinellosis in my country, which has the advantages of simple operation, etc., but requires the quarantine personnel to have good professional knowledge and experience, and the labor intensity of the staff is high. In addition, meat infection It is easy to miss detection when the intensity is low
PCR diagnostic technology has greatly improved the sensitivity of inspection and quarantine methods. However, due to unfavorable factors such as the high price of PCR instruments and the need for electrophoresis analysis of amplified products, the application of this method is greatly restricted.

Method used

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  • A rapid detection method and primer composition of trichinella spiralis based on lamp technology
  • A rapid detection method and primer composition of trichinella spiralis based on lamp technology
  • A rapid detection method and primer composition of trichinella spiralis based on lamp technology

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Example 1. Establishment of Trichinella spiralis LAMP detection method

[0053] 1. DNA extraction from muscle larvae of Trichinella spiralis

[0054] (1) ICR mice were sacrificed after being artificially infected with Trichinella spiralis for 35 days, and the diaphragm, masseter, tongue and limb muscles were taken and cut into 1-2 mm square meat pellets.

[0055] (2) Prepare artificial pepsin digestion solution (1% pepsin, 1% concentrated hydrochloric acid), put the above mouse muscle fragments in an Erlenmeyer flask, add 20 ml of digestion solution per gram of sample, and incubate at 37°C for 4 hours , set the rotation speed of the Erlenmeyer flask to 100rpm.

[0056] (3) Filter the digested solution with a nylon sieve, let the filtrate stand for 20 minutes, remove the supernatant, take the muscle larvae of Trichinella spiralis in the sediment, and observe it under a microscope.

[0057] (4) According to the instructions of the Omega kit, the total DNA of the muscle ...

Embodiment 2

[0080] Example 2. Application of Trichinella spiralis LAMP detection method

[0081] 1. Detection of pork samples

[0082] (1) Apply the LAMP technology established by the present invention to the detection of 70 pig tongue samples. Get porcine tongue muscle 1-2g, according to the operation 1 DNA extraction method among the embodiment 1, extract the total DNA of muscle sample, detect with conventional PCR and LAMP method respectively (see operation 2 and 3 in the embodiment 1).

[0083] (2) The result is as follows Figure 5 As shown, it was found that after LAMP amplification, the positive control had ladder-like bands, and the reaction product was green after staining, while all pork samples had no ladder-like bands (negative), and all samples were orange (negative). The routine PCR test results were consistent with the LAMP test results.

[0084] 2. Beef sample testing

[0085] (1) Apply the LAMP technology established by the present invention to the detection of 10 bee...

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Abstract

The invention discloses a method and a primer combination for quickly detecting trichinella spiralis on the basis of LAMP (loop-mediated isothermal amplification) technologies. The LAMP primer combination for detecting the specificity of the trichinella spiralis comprises a primer F3, a primer B3, a primer FIP and a primer BIP. Sequences of the primers F3, B3, FIP and BIP are respectively shown as SEQ ID NO.1-SEQ ID NO.4. The primer combination can be applied to preparing reagents for detecting the trichinella spiralis. The method and the primer combination have the advantages that the primer combination and the corresponding method are high in specificity and sensitivity; different pathogens such as the trichinella spiralis, Escherichia coli, cysticerus cellulosae and toxoplasma gondii can be detected, and results can and only can be positively amplified from trichinella spiralis samples; the sensitivity of the method is 1000 times the sensitivity of common PCR (polymerase chain reaction) detection technologies, and the trichinella spiralis can be detected even if a gram of meat products only contains one muscle larva.

Description

technical field [0001] The invention belongs to the technical fields of veterinary biotechnology and molecular biology, and relates to a rapid detection method and a primer composition of Trichinella spiralis based on LAMP technology. Background technique [0002] Trichinellosis is a worldwide zoonotic parasitic disease, which is prevalent among mammals, and humans and animals are infected by eating raw or semi-cooked meat containing Trichinella cysts. The disease is very harmful, not only causing huge economic losses to animal husbandry production, but also posing a serious threat to human health. my country is one of the few countries where the disease is most harmful in the world, and it has been listed as the first of the three zoonotic parasitic diseases (trichinellosis, cysticercosis and echinococcosis), and as The import and export of meat, the slaughter of animals, and the zoonotic diseases that the Chinese government proposes to allow people to eat "safe meat" are t...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12Q1/6844C12Q1/6888C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2563/107C12Q2565/125
Inventor严若峰李祥瑞徐立新宋小凯黄芸
OwnerNANJING AGRICULTURAL UNIVERSITY