CN104059974A - Pig trichina detection kit based on 18 SrRNA (ribosomal Ribose Nucleic Acid) and application of pig trichina detection kit - Google Patents

Abstract

The invention provides a pig trichina detection kit based on 18 SrRNA (ribosomal Ribose Nucleic Acid) and an application of the pig trichina detection kit. The pig trichina detection kit consists of aseptic deionized water, PCR (Polymerase Chain Reaction) liquid, TaqDNA (Taq Deoxyribose Nucleic Acid) Polymerase, GoldView DNA dye, a bromophenol blue sample-loading buffer solution, a standard substance and a reference substance. The kit is capable of quickly and accurately detecting the pig trichinae DNA in a detected sample and can also be used for molecular epidemiology research and treatment effect monitoring on pig trichina infection. The kit is simple in operation step, low in cost and especially capable of saving time, saving labor and saving a large quantity of reagents and material consumption when the kit is used for large-scale detection of a large quantity of samples, so that the working efficiency is improved. A method using the kit is a detection method which is high in sensibility and strong in specificity on the basis of molecular biology. Through the kit, the detection accuracy rate is greatly increased and the false positive rate is reduced; the kit can be used for detecting the pig trichinae.

Description

Trichinella sui detection kit and application based on 18S rRNA gene

Technical field

The invention belongs to biotechnology, relate in particular to the parasitic test kit of this food of a kind of detection Trichinella sui source property, can in differentiating the sample that detects infected pigs Trichinella spiralis, apply.

Background technology

Trichonematosis is a kind of zoonosis of serious harm public health security, it be by Trichinella spiralis ( trichinella spiralis) adult parasitizes Mammals enteron aisle and parasitized larvae in the caused disease of muscle tissue.

Trichonematosis is worldwide distribution, is wherein no lack of developed country, and China is one of the most serious several countries of Trichinella spiralis harm in the world.The host range that Trichinella spiralis can infect is very extensive, humans and animals is all had significantly pathogenic, and in domestic animal, except the carnivores such as cat and dog, pig morbidity is the highest.Can be throughout the year parasitic in host after trichinzation, infected animal is with worm throughout one's life.But ill pig does not often have clinical symptom, the mankind are because eating half-mature or raw infecting containing trichinous pork raw, the symptom that usually occurs gastrointestinal symptom, heating, oedema, blood eosinophilia, agnogenic long-term sore muscle etc., severe one disability, even dead.Because trichinous clinical manifestation is comparatively complicated, animal atypical symptoms when infecting early stage or low-grade infection especially, often cause and ignore or mistaken diagnosis, thereby incur loss through delay treatment opportunity and cause heavy losses, so Accurate Diagnosis trichonematosis in time treatment are to control relatively effective means of this disease.Therefore, control trichinous popular must first picking up from source, strictly control food-borne parasitic disease popular in livestock and poultry, hold and butcher quarantine pass.

Yet at present food safety and sanitation problem is very severe, but it is quite backward to butcher Quarantine Techniques.Sediments microscope inspection method when the trichinous detection of slaughtered animals still adopts and found this parasite before approximately 180 years, very easily causes undetected.This has had a strong impact on meat quality, causes grave danger also to human consumer's health simultaneously.Therefore, imperative to setting up a kind of simple to operate, quick, responsive, special detection Trichinella sui method.PCR method be a kind of simple to operate, susceptibility is high, the molecular biology for detection of high specificity, it can be in a reaction system goal gene fragment of DNA amplification sample, be specially adapted to the quick diagnosis of a large amount of clinical samples of infecting.Therefore, set up Trichinella spiralis PCR method for quick, the scientific basis that provides of preventing and treating clinically this disease is provided, and to improving detection, the monitoring level of animal epidemic, ensure that animal food safety is significant.

Summary of the invention

The object of this invention is to provide a kind of Trichinella sui detection kit based on 18S rRNA gene, this test kit comprises: aseptic deionized water, PCR reaction solution, taqarchaeal dna polymerase, GoldView DNA dyestuff, tetrabromophenol sulfonphthalein sample-loading buffer, standard substance, reference substance.

Wherein, PCR reaction solution is by PCR damping fluid, MgCl ₂, dNTPs, detection form with primer, detect with primer and be respectively and detect totally 2 of the upstream primer of Trichinella sui and downstream primers:

Trichinella sui upstream primer (SEQ ID No:1): 5 ' -cATTCGTGGTTGCTGTTGTTGC-3 ',

Trichinella sui downstream primer (SEQ ID No:2): 5 ' -cGTTGACGCTTTCATTGTAG-3 ',

Standard substance for have Trichinella sui ( trichinella spiralis) recombinant plasmid, its nucleotide sequence is as shown in SEQ ID No:3.

Reference substance is divided into positive reference substance and negative control product, and positive control is the DNA sample that has Trichinella sui DNA fragmentation, and negative control is the DNA sample without Trichinella sui DNA.

This test kit is stored in-20 ℃, reduces multigelation as far as possible.

Another object of the present invention is to provide the application of described test kit in detecting this food of Trichinella sui source property parasite.

test kit using method of the present invention:

Each detection all should be set up positive control and negative control.Standard substance are 60 ng/ μ l with aseptic deionized water dilution.

1. PCR reaction tubes preparation: cumulative volume is 25 μ l, PCR reaction solution is in melting on ice and preparing PCR reaction tubes.Get successively PCR reaction solution 22.8 μ l, taqarchaeal dna polymerase 0.2 μ l, the sample DNA of concentration 60 ng/ μ l (sample to be detected, standard substance, positive reference substance, negative control product) 2 μ l mix in PCR reaction tubes.With finger, flick the PCR reaction tubes preparing or with micropipet piping and druming, mix instantaneous high speed centrifugation 2 s.

2. augmentation detection: PCR reaction tubes is placed on PCR instrument, reaction conditions is set is: 95 ℃ of denaturation 5 min, 94 ℃ of sex change 45 s, 60 ℃ of annealing 45 s, 72 ℃ extend 60 s totally 30 circulations, 72 ℃ are extended 8 min, 4 ℃ of preservations.

3. detected result is judged: 1 g agarose is dissolved in 0.5 * TBE electrophoretic buffer of 100 ml, then adds 5 μ l GoldView DNA dyestuffs, after mixing, boil, topple over gel slab.After gel cooled and solidified, PCR product 8 μ l are mixed with appropriate sample-loading buffer, add and in gel pore, carry out electrophoresis.Electrophoresis finishes rear taking-up gel and is placed in observation under ultraviolet lamp, takes pictures.Standard substance hole and positive control hole all have object band and negative control hole without the success of any band explanation PCR reaction detection.Electrophoretic band and the standard substance hole that tested sample aperture occurs, positive control hole respective strap is in the same size is judged to be the positive, otherwise negative.

Advantage of the present invention: the invention provides a kind of Trichinella sui substance PCR detection kit, its advantage is that a reaction can detect the Trichinella sui DNA in tested sample quickly and accurately, also can be used for Molecule Epidemiology Investigation and curative effect monitoring that Trichinella sui infects.The simple expense of template DNA step of present method is low, during in particular for the extensive detection of a large amount of samples, not only time saving and energy saving but also saved a large amount of reagent and consumptive material, has improved working efficiency.Present method is to be based upon that a kind of susceptibility on molecular biology mechanism is high, the detection method of high specificity, has greatly improved Detection accuracy, has reduced false positive rate.

Accompanying drawing explanation

The PCR product electrophoresis result of Fig. 1 Trichinella sui standard substance.

The PCR detected result of Fig. 2 Trichinella sui positive sample.

Fig. 3 the present invention is to the part detection result of specimen gathering.

Embodiment

The present invention is described further with specific embodiment by reference to the accompanying drawings.Should be appreciated that, these embodiment are only for illustration purpose, and are not used in the restriction scope of the invention.

embodiment mono-

The invention provides a kind of Trichinella sui PCR detection kit based on 18S rRNA gene, this test kit comprises: aseptic deionized water, PCR reaction solution, taqarchaeal dna polymerase, GoldView DNA dyestuff, tetrabromophenol sulfonphthalein sample-loading buffer, standard substance, reference substance.

Wherein, PCR reaction solution is by PCR damping fluid, MgCl ₂, dNTPs, detection form with primer, detect with primer and be respectively and detect the upstream primer of Trichinella sui and downstream primer totally 2 (table 1).

Standard substance for have Trichinella sui ( trichinella spiralis) recombinant plasmid, its nucleotide sequence is as shown in SEQ ID No:3.

Reference substance is divided into positive reference substance and negative control product, and positive control is the DNA sample that has Trichinella sui DNA fragmentation, and negative control is the DNA sample without Trichinella sui DNA.This test kit is stored in-20 ℃, reduces multigelation as far as possible.

the PCR of embodiment bis-Trichinella sui standard substance detects

1. material

taqdNA polymerase reaction system, pMD18-T cloning system, general genome extract test kit (Universal Genomic DNA Extraction Kit Ver. 3.0), DNA molecular amount standard marker all purchased from precious biotechnology (Dalian) company limited, plasmid in a small amount extraction agent box, gel to reclaim test kit be the Hangzhou Chinese mugwort company limited's product of pursuing progress, the common agents such as penbritin are all purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and PCR instrument is German Biometra T-gradient PCR instrument.

2. design of primers and synthetic (table 2).

The above-mentioned worm kind goal gene sequence of take is template, uses Primer Premier 5.0 softwares to analyze, design primer, by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, is synthesized.

3. examination criteria product preparation

Get respectively the polypide of Trichinella sui, according to general genome, extract test kit specification sheets and extract genomic dna as template, with the standard substance upstream and downstream primer of corresponding polypide, on PCR instrument, increase, reaction conditions is 95 ℃ of denaturation 5 min, 94 ℃ of sex change 45 s, 60 ℃ of annealing 45 s, 72 ℃ extend 60 s totally 30 circulations, 72 ℃ are extended 8 min, 4 ℃ of preservations.PCR product cuts object band and carries out gel recovery after 1% agarose electrophoresis confirmation stripe size is correct.Reclaim product and insert pMD18-T carrier with cloning system, and be transformed into intestinal bacteria cultivating containing in the substratum of penbritin, send Hangzhou Genomics R & D Center to carry out sequence verification positive colony.Order-checking shows that the correct corresponding gel recovery product of Insert Fragment is adjusted into 60 ng/ μ l with aseptic deionized water and is standard substance.

4. result

Through order-checking, show, above-mentioned standard product completely and expection meet, Trichinella sui standard substance goal gene sequence is respectively as the SEQ ID No:3 of sequence table.PCR result is referring to Fig. 1: M, DNA molecular amount standard marker; 1: Trichinella sui substance PCR standard substance band; 2: negative control product.

the PCR of embodiment tri-Trichinella sui positive sample detects

1. specimen dna extracts

Gathering respectively 9 grams of the positive pig liver samples of Trichinella sui that 4 examples make a definite diagnosis through traditional method, by general genome DNA extracting reagent kit specification sheets, extract DNA, is 60 ng/ μ l by aseptic deionized water adjustment DNA concentration.

2. Samples detection

Take out PCR reaction solution and get 22.8 μ l after ice bath melts, add archaeal dna polymerase 0.2 μ l, then add specimen dna 2 μ l to be checked.3 PCR reaction tubess are set in addition again, add successively mentioned reagent, but DNA replaces with standard substance, positive reference substance, negative control product respectively simultaneously.The effective micropipet piping and druming of PCR reaction reagent preparing mixes rear instantaneous high speed centrifugation 2 s, the following reaction conditions of operation after being placed on PCR instrument: 95 ℃ of denaturation 5 min, 94 ℃ of sex change 45 s, 60 ℃ of annealing 45 s, 72 ℃ extend 60 s totally 30 circulations, 72 ℃ are extended 8 min, 4 ℃ of preservations.

3. detected result

Detected result is referring to Fig. 2, S in figure: standard control; P: positive control; N: negative control; 1: Trichinella sui is positive.

the application of embodiment tetra-Trichinella sui detection kit

Method is with embodiment tri-, and random acquisition pig liver sample extracts DNA, pcr amplification, and result is referring to Fig. 3: Far Left two swimming lanes " P, N " are respectively positive control and negative control; 3,11 and 18 swimming lanes are that Trichinella sui is positive; All the other swimming lanes are all negative.

The present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally.

<110> Zhejiang University

Trichinella sui detection kit and the application of <120> based on 18S rRNA gene

<160>3

<170>DNAStar5.0

<210>1

<211>22

<212>DNA

<213> Trichinella sui (Trichinella spiralis)

<220>

<221>RRNA

<400>1

cattcgtggt tgctgttgtt gc 22

<210>2

<211>20

<212>DNA

<213> Trichinella sui (Trichinella spiralis)

<220>

<221>RRNA

<400>2

cgttgacgct ttcattgtag 20

<210>3

<211>798

<212>DNA

<213> Trichinella sui (Trichinella spiralis)

<220>

<221>RRNA

<400>3

cattcgtggt tgctgttgtt gctcttcatt gagtgtcaat ggtgcctcga gattttactt 60

tgaaaaaatt agagtgctca aagcaggttg tgatgcctga ataatggtgc atggaataat 120

agaatacgat ctcggttcta ttttgttggt tttcgaaccg gagataagta ttgaaaggaa 180

cagacggggg cattcgtatt gctgcgttag aggtgaaatt cttggatcgc agcaagatga 240

acaattgcga aagcatttgc caagaatgtt ttcattaatc aagaacgaaa gttagaggtt 300

cgaaggcgat cagataccgc cctagttcta acggtaaact atgccaacca gcgattcgcc 360

gaagttcatt taagactcgg cgagcagctt ccgggaaacc aaagtgtttc ggttccgggg 420

gaagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca ccaggagtgg 480

agcctgcggc ttaatttgac tcaacacggg aaaactcacc cggcccggac actggaagga 540

ttgacagatt gacagctctt tcttgattca gtgggtagtg gtgcatggcc gttcttagtt 600

ggtggagcga tttgtctggt taattccgat aacgaacgag actctaccct attaaatagt 660

gacagtattg tattttttgt gctgatcact tcttagaggg actggcaaat tgaaagctgc 720

acgaaaaaga gcaataacag gtctgtgatg cccttcgatg tccggggctg cacgcgcgct 780

aaaatgaaag cgtcaacg 798

Claims (3)

1. the Trichinella sui detection kit based on 18S rRNA gene, is characterized in that, this test kit by aseptic deionized water, PCR reaction solution, taqarchaeal dna polymerase, GoldView DNA dyestuff, tetrabromophenol sulfonphthalein sample-loading buffer, standard substance, reference substance form, and wherein, PCR reaction solution is by PCR damping fluid, MgCl ₂, dNTPs, detection form with primer, detect and be respectively with primer:

Trichinella sui upstream primer: 5'-CATTCGTGGTTGCTGTTGTTGC-3',

Trichinella sui downstream primer: 5'-CGTTGACGCTTTCATTGTAG-3',

Standard substance for have Trichinella sui ( trichinella spiralis) recombinant plasmid, its nucleotide sequence as shown in SEQ ID No:3,

Reference substance is divided into positive reference substance and negative control product, and positive control is the DNA sample that has Trichinella sui DNA fragmentation, and negative control is the DNA sample without Trichinella sui DNA.

2. a kind of Trichinella sui detection kit based on 18S rRNA gene according to claim 1, is characterized in that, test kit is stored in-20 ℃, reduces multigelation as far as possible.

3. the application of test kit according to claim 1 in detecting food source property parasite Trichinella sui.