CN103255226A - Trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as kit and detection method - Google Patents

Abstract

The invention discloses a trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as a kit and a detection method. In allusion to a gene sequence of the trichina, detection primers SEQ ID NO.1-2 are designed and a PCR-DHPLC method is used for the qualitative detection of the trichina in detected animal derived food. The kit comprises Taq DNA polymerase with concentration of 5U/microlitre, 2.5mM of PCR reaction liquid, 2.5mM of dNTP (Diethyl-Nitrophenyl Thiophosphate), 10 microns of SEQ ID NO.1 and 10 microns of SEQ ID NO.2. The method disclosed by the invention can be used for precisely detecting the polypide sample of one trichina, and has the advantages of being short in detection time and simple to operate, saving lots of manpower and material resources and being suitable for rapid detection.

Description

Trichinella spiralis PCR-DHPLC detects primer, test kit and detection method

Technical field

The present invention relates to the detection method of Trichinella spiralis in the animal derived food, relate in particular to the method for utilizing Trichinella spiralis in PCR and sex change high performance liquid chromatography (DHPLC) the technology for detection animal derived food.Also relate to and detect employed composition, i.e. test kit.

Background technology

Harmful organism in food and the agricultural byproducts mainly comprises bacterium, virus and parasite, among the Amphixenosis according to World Health Organization's announcement in 1999, someone is total to ill 58 kinds by beastly parasite, investigates ill 91 kinds altogether of the beastly parasites of Chinese people in year February in April, 2004-2005 according to The Fourth Military Medical University of PLA.The people beast suffers from parasitosis altogether, and public health, people ' s health, social stability are all brought serious threat and harm.

Hair shape nematode is called for short Trichinella spiralis, belongs to the first order of hair, Trichnellidae, hair shape genus.Its life history is special, and as intermediate host and final host, the small intestine that adult colonizes in the host claims the intestines Trichinella spiralis with same host, and each muscle that larva parasitizes the host claims the flesh Trichinella spiralis.Mammalss such as people, pig, dog are its host, colonize in duodenum and jejunum front portion, and adult is attached to the intestines wall.The larva viviparity is distributed to whole body through blood, lymph, only just continues to grow in voluntary muscle, forms packing.Ripe packing is infected by the host back of eating, and larva is through 4 decortications, grows to be adult.

Trichinosis is one of important parasite disease of infecting both domestic animals and human, and its host carnivore, muroid, can infect except pig per capita.Think in the domestic animal that the poultry pig is main, but causing death during infected person.Trichinella spiralis is distributed in all over the world, wide-scale distribution between Mammals, and its major cause has: host animal is of a great variety, and it is wide to distribute, and natural epidemic disease source is abundant; The resistibility of larva is very strong in the muscle packing, can survive 57 days at-20 ℃, can survive more than 100 days at the muscle of corruption, and salt marsh and sootiness all can not be killed the larva of deep part of muscle; Pig, mouse all are omnivores, it is generally acknowledged the pig that infects Trichinella spiralis how to eat well dead mouse, fly maggot, wash meat water and meat bits etc.The scope of activity of dog is big, and the chance of having carcase is many, and the situation of infection is serious.Therefore, propagating mutually between pig, mouse, dog and the people is the popular key point of this disease.

China still is in the relatively backward stage to the detection technique of Trichinella spiralis in the food, the method of inspection also is only limited to traditional morphologic detection, mainly be to use visual method, compressing tablet sediments microscope inspection method, dyeing pressed disc method and digestion method, detect the Trichinella spiralis in the animal derived foods such as pork and beef.Detect complex steps, method sensitivity is low; The result judges main according to the morphology check, requires high and be subject to subjective experience to influence for reviewer's professional ability.Adopt the ELISA method to detect parasitic ovum, poor specificity, be prone to false positive.That the PCR detection technique has is accurate, quick, highly sensitive, good reproducibility, stable advantages of higher.This method remedied the ordinary method sense cycle long, loaded down with trivial details, be subject to deficiencies such as human factor influence.Though it is low that the PCR-gel electrophoresis detection method detects cost, the reaction product electrophoretic process very easily pollutes; High specificity, highly sensitive real time fluorescent PCR method also exists the detection cost higher, and the fluorescent probe shelf time is than problems such as weak points.

Summary of the invention

The present invention uses PCR to carry out Trichinella spiralis detection in the animal derived food in conjunction with the DHPLC technology.Sex change high performance liquid chromatography (DHPLC) adopts the principle of ion pair RPLC, adopts the mode of temperature adjusting simultaneously by using special high temperature resistant liquid phase chromatography column, and the nucleotide fragments molecule is carried out analytical separation.Its fast high-flux, full-automatic operation, accuracy height, good reproducibility and the high advantage of susceptibility make it have the advantage that can not be substituted in foranalysis of nucleic acids.Detect with traditional Trichinella spiralis and detection methods such as authentication method such as morphology evaluation, immunological technique, PCR-gel electrophoresis are compared, method and test kit that the present invention sets up, greatly shortened the time of operation, and reduce the appearance of false positive and false negative result greatly, make the result more accurately, reliable.

Technical scheme of the present invention is: extract sample DNA, be template with DNA, adopt the specific detection primer of Trichinella spiralis to carry out pcr amplification respectively, gather the figure spectrum information of goal gene again with the DHPLC technology, directly read the detected result of Trichinella spiralis from the figure spectrum information, on this basis, the present invention has also carried out specificity and sensitivity detection to this method; Its concrete operations step is as follows:

An aspect of of the present present invention is: provide a kind of Trichinella spiralis to detect primer, be specially following base sequence: upstream primer, SEQ ID NO.1; Downstream primer, SEQ ID NO.2.

Another aspect of the present invention is: a kind of Trichinella spiralis detection kit is provided, and it comprises primer SEQ ID NO.1～2 mentioned above.

For the Trichinella spiralis detection kit described in the technique scheme, comprise that concentration is Taq archaeal dna polymerase and the PCR reaction solution of 5U/ μ L; Independent packaging respectively; Wherein, described PCR reaction solution is mixed solution, wherein contains outside each 10 μ M of primer SEQ ID NO.1 mentioned above～2, also comprises 10mM TrisHCl, 50mM KCl, 25mM MgCl ₂, each 2.5mM of dNTP.Test kit preservation condition :-20 ℃ of preservations.

Another aspect of the present invention is: the detection method of Trichinella spiralis in a kind of animal derived food is provided, and it utilizes the Trichinella spiralis primer detection kit described in the technique scheme, sample DNA is carried out PCR method detect.

For the detection method of Trichinella spiralis in the animal derived food described in the technique scheme, it comprises following operation steps: respectively sample is carried out PCR as follows and detect:

1. the PCR reaction system is 25 μ L, wherein:

Sample DNA 2 μ L

5U/ μ L Taq archaeal dna polymerase 0.2 μ L

PCR reaction solution 12 μ L

Water 10.8 μ L

2. reaction conditions: 94 ℃ of pre-sex change 5min; Enter circulation: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 40s, 40 circulations; 72 ℃ stop extending 10min.

Detection method for Trichinella spiralis in the animal derived food described in the technique scheme, in its operation steps, can also comprise that DHPLC detects, its principle of work is: DHPLC adopts the high-pressure closing liquid-phase flow path, the DNA sample is injected and flows through DNA sample separation post to be measured automatically under damping fluid carries, by the different graded of damping fluid, under different separator column temperature condition, realize the different analysis of DNA sample to be measured; By ultraviolet detection or the separated DNA sample of fluoroscopic examination.

The PCR product that the PCR detection method of Trichinella spiralis mentioned above is obtained carries out DHPLC to be analyzed, and condition is as follows:

Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;

Column temperature: 50 ℃;

Moving phase: 0bp, 55.0% buffered soln A, 45.0% buffered soln B,

100bp, 50.2% buffered soln A, 49.8% buffered soln B,

300bp, 39.4% buffered soln A, 60.6% buffered soln B,

500bp, 36.0% buffered soln A, 64.0% buffered soln B,

700bp, 34.3% buffered soln A, 65.7% buffered soln B,

900bp, 33.3% buffered soln A, 66.7% buffered soln B, wherein,

Buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;

Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;

Flow velocity: 0.9mL/min;

Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;

Applied sample amount: PCR product 10 μ L.

After detecting end, test sample does not have the amplification absorption peak and occurs, and can judge that sample result is negative, and directly report does not detect Trichinella spiralis; If typical PCR product absorption peak appears in test sample, and absorption peak can judge that this sample result is positive during greater than 3mV; Typical PCR product absorption peak appears in test sample, but absorption peak is during less than 3mV, and the suggestion sample is reformed.The results peaks of reforming absorption value is still then negative less than 3mV, otherwise is probable positive.

Beneficial effect: use detection kit of the present invention and detection method, can detect Trichinella spiralis in the animal derived food in high sensitivity, and detect weak point consuming time, simple to operation, can save a large amount of labour's material resources, be fit to the requirement of rapid detection.Succeeding in developing and applying of this method detects cost to improving detection efficiency and the sensitivity of Trichinella spiralis in the food, reducing, and is with a wide range of applications to carrying out food safety monitoring and improving inspection technology.

Description of drawings

Accompanying drawing 3 width of cloth of the present invention are respectively:

Fig. 1 is the PCR-DHPLC detected result of embodiment 1;

Fig. 2 is the specificity test-results spectrogram of embodiment 2; 1-8 is followed successively by: Trichinella spiralis, cysticercus, roundworm egg, whipworm, hookworm, liver fluke, fasciloopsis, trichostrongyle;

Fig. 3 is the sensitivity test spectrogram as a result of embodiment 3; 1,5 Trichinella spiralis samples; 2,1 Trichinella spiralis sample.

In the above-mentioned accompanying drawing 1～3, X-coordinate is retention time, and (unit: minute min), ordinate zou is represented absorption peak strength of signal (unit: millivolt mV).

Embodiment

Following non-limiting example, its foundation and application thereof to present method is further described, and can make those of ordinary skill in the art more fully understand the present invention, but not limit the present invention in any way.

If no specified otherwise, the employed main agents in this part, instrument and merchandise resources thereof are: reagent such as Taq enzyme and PCR damping fluid are all available from precious biotechnology (Dalian) company limited; Triethylamine acetyl salt (TEAA, chromatographically pure) is available from Transgenomic company; Acetonitrile (chromatographically pure) is available from Fisher company; Regular-PCR instrument PE24000(PerkinElmer company, the U.S.); Sex change high performance liquid chromatograph NAV-99-4500 (Transgenomic company, the U.S.); Supercentrifuge centrifuge 5804(Eppendorf company, Germany).

Institute of the present invention derives from Shanghai parasite institute of Chinese Academy of Agricultural Sciences livestock and poultry important parasite worm kind resources bank with Trichinella spiralis, cysticercus, roundworm egg, whipworm, hookworm, liver fluke, fasciloopsis, trichostrongyle; Jilin Agriculture University and parasite teaching and research room of Dalian Medical Univ.

Embodiment 1

(1) assembling of primer design, synthetic and test kit:

Primer sequence and expanding fragment length that present embodiment is identified for detecting are as follows:

Upstream primer: 5 '-gcg aat tct tgg atc gga gac ggc ctg-3 '

Downstream primer: 5 ′ – gct cta gac gag atg tcg tgc ttt caa cg-3 '

Trichinella spiralis amplifies 740bp specificity target gene fragment.

Be designed for the test kit that PCR-DHPLC detects on this basis.Comprise in this test kit that concentration is Taq archaeal dna polymerase and the PCR reaction solution of 5U/ μ L; Contain 10mM TrisHCl, 50mM KCl, 25mM MgCl in the PCR reaction solution wherein ₂, dNTP(dATP, dGTP, dCTP and dTTP) each 2.5mM and Trichinella spiralis detect primer to 10 μ M.

(2) foundation of PCR-DHPLC detection method:

The detection kit that this detection method uses present embodiment to set up comprises the steps:

1) specimen preparation

1. identify suspicious Trichinella spiralis sample for morphology, with scissors clip 100mg polypide tissue (can from different positions clip), shred tissue as far as possible, put into the 1.5mL centrifuge tube.

2. treat the sample product: peel off the muscle of 100mg Suspected Area, do not have bone in the muscle of separation, be cut into the cube meat of about 0.5cm3 size; Be put in the tissue homogenizer, add a little gastric pepsin digestion liquid after, 1200g, homogenate 3min.

2) preparation of template DNA

1. in the 1.5mL centrifuge tube of sample is housed, add 400 μ L DNA extraction buffers and 40 μ L Proteinase Ks (10mg/mL), fully digest 3h down at 37 ℃ behind the mixing;

2. the centrifugal 2min of 10000g abandons supernatant liquor; Add isopyknic phenol, chloroform, primary isoamyl alcohol (25:24:1), mix organic phase and water by thermal agitation, 10000g, 4 ℃ of centrifugal 10min shift supernatant in another centrifuge tube.

3. isopyknic supernatant of getting adds chloroform, primary isoamyl alcohol (24:1) vibration, centrifugal 12000g, 10min; Extracting once.

4. get 2 times of volume of ethanol (or adding isopyknic Virahol) of supernatant adding precooling, the high vibration mixed solution ,-20 ℃ of precipitations are spent the night or-80 ℃ of precipitation 30min; 12000g, centrifugal 10min collects the DNA precipitation; Carefully abandon supernatant, centrifuge tube is inverted on the thieving paper, liquid is blotted, add 1mL70% washing with alcohol precipitation; 10000g, centrifugal 2min; Carefully abandon supernatant, centrifuge tube is placed on the super clean bench dry up.

5. use 50 μ L TE dissolving DNA again, gentle vibration.The DNA that extracts is standby in-20 ℃ of preservations.

3) pcr amplification

Get 2 μ L testing sample dna solutions, add PCR reaction solution, 0.2 μ L Taq archaeal dna polymerase and sterilization ultrapure water 10.8 μ L in the 12 μ L test kits, cumulative volume 25 μ L; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:

Pre-sex change: 95 ℃, 5min;

Enter circulation: 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 40s, 40 circulations;

Stop extending: 72 ℃, 15min

4 ℃ of preservations of PCR product;

4) DHPLC analyzes

The PCR product is carried out DHPLC analyze, the DHPLC analysis condition is as follows:

Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;

Column temperature: 50 ℃;

Moving phase (volume ratio): 0bp, 55.0% buffered soln A, 45.0% buffered soln B,

100bp, 50.2% buffered soln A, 49.8% buffered soln B,

300bp, 39.4% buffered soln A, 60.6% buffered soln B,

500bp, 36.0% buffered soln A, 64.0% buffered soln B,

700bp, 34.3% buffered soln A, 65.7% buffered soln B,

900bp, 33.3% buffered soln A, 66.7% buffered soln B,

Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;

Flow velocity: 0.9mL/min;

Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;

Applied sample amount: PCR product 10 μ L.

The experimental result of present embodiment is with collection of illustrative plates form output in the description of drawings, the PCR-DHPLC detected result as shown in Figure 1: typical PCR product absorption peak appears in test sample, and absorption peak judges that greater than 3mV this sample result is positive, detects Trichinella spiralis.

The test of embodiment 2 specificitys

Get parasite samples such as Trichinella spiralis, cysticercus, roundworm egg, whipworm, hookworm, liver fluke, fasciloopsis, trichostrongyle, extract genomic dna according to embodiment 1 described method.Be template with these genomic dnas, carry out pcr amplification and DHPLC detects according to embodiment 1 described method, result 1-8 as shown in Figure 2 is followed successively by: Trichinella spiralis, cysticercus, roundworm egg, whipworm, hookworm, liver fluke, fasciloopsis, trichostrongyle; Have only the Trichinella spiralis sample typical PCR product absorption peak to occur, absorption peak judges that greater than 3mV this sample result is positive, detects Trichinella spiralis.Parasite samples such as other cysticercus, roundworm egg, whipworm, hookworm, liver fluke, fasciloopsis, trichostrongyle are all negative, do not have false positive and false negative result.Illustrate and adopt PCR-DHPLC method of the present invention to detect the Trichinella spiralis high specificity.

The test of embodiment 3 detection sensitivities

Microscopically counting Trichinella spiralis polypide, distinguish 5 Trichinella spiralis bodies of extracting and 1 Trichinella spiralis body DNA according to the method that embodiment 1 sets up, respectively get 2 μ L as template, carry out the PCR-DHPLC detection according to the method that embodiment 1 sets up, the result is as shown in Figure 3: get after 1 Trichinella spiralis polypide extracts DNA, the template DNA that adds 2 μ L still can detect typical positive absorption peak through PCR-DHPLC.This result shows that the sensitivity of present method is very high, can detect the single tissue DNA of Trichinella spiralis.

The practical application of embodiment 4 methods

During 10 months of year June in August, 2011 to 2012, Trichinella spiralis PCR-DHPLC detection kit and detection method in the animal derived food that employing embodiment 1 sets up, adopt traditional digestion method and compressing tablet sediments microscope inspection morphological method simultaneously, detect actual sample.Wherein detect 27 parts of porks, two kinds of methods all detect Trichinella spiralis in 2 duplicate samples; Detect 15 parts in mouse sample, two kinds of methods all detect Trichinella spiralis in 1 duplicate samples; Detect 21 parts in dog meats sample, two kinds of methods all do not detect Trichinella spiralis; Detect go down to posterity 17 of mouse of Trichinella spiralis, two kinds of methods all detect Trichinella spiralis in 17 duplicate samples.The result shows: Trichinella spiralis and morphologic detection result's degree of agreement is fine in the application PCR-DHPLC method test sample.

Table 1 practical application detected result

In the present embodiment, the digestion method of use and compressing tablet sediments microscope inspection morphological method detect each sample average (comprising specimen preparation) consuming time 28h always, comprising sample preparation digestion 24h, detect 4h consuming time, detect consuming time, complex operation step; Use the PCR-DHPLC method, detect each sample average (comprising specimen preparation) consuming time 6h always, detect (not comprising specimen preparation) consuming time 2.5h, easy and simple to handle quick.

Claims (6)

1. a Trichinella spiralis PCR-DHPLC detects primer, it is characterized in that comprising base sequence: upstream primer SEQ ID NO.1; Downstream primer SEQ ID NO.2.

2. a Trichinella spiralis PCR-DHPLC detection kit is characterized in that: comprise the described primer SEQ of claim 1 ID NO.1～2.

3. Trichinella spiralis PCR-DHPLC detection kit according to claim 2 is characterized in that: comprise that concentration is Taq archaeal dna polymerase and the PCR reaction solution of 5U/ μ L;

Wherein, described PCR reaction solution is by the described primer SEQ of claim 1 ID NO.1～2 each 10 μ M, TrisHCl10mM, KCl 50 mM, MgCl ₂Each 2.5 mM of 25 mM, dNTP form.

4. the PCR-DHPLC detection method of Trichinella spiralis in the animal derived food is characterized in that: comprise and utilize the described Trichinella spiralis detection kit of claim 2, sample DNA is carried out PCR method detect.

5. the PCR-DHPLC detection method of Trichinella spiralis in the animal derived food according to claim 4 is characterized in that: describedly sample DNA is carried out the step that PCR method detects comprise:

1. the PCR reaction system is 25 μ L, wherein:

Sample DNA 2 μ L

5U/ μ L Taq archaeal dna polymerase 0.2 μ L

PCR reaction solution 12 μ L

Water 10.8 μ L

2. reaction conditions: 94 ℃ of pre-sex change 5min; Enter circulation: 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 40s, 40 circulations; 72 ℃ stop extending 10min.

6. the PCR-DHPLC detection method of Trichinella spiralis in the animal derived food according to claim 5 is characterized in that: also comprise following operation steps:

The PCR product that the described method of claim 5 is obtained carries out the DHPLC analysis, and condition is as follows:

Chromatographic column: PS-DVB ﹠amp; The C18DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;

Column temperature: 50 ℃;

Moving phase: 0bp, 55.0% buffered soln A, 45.0% buffered soln B,

100bp, 50.2% buffered soln A, 49.8% buffered soln B,

300bp, 39.4% buffered soln A, 60.6% buffered soln B,

500bp, 36.0% buffered soln A, 64.0% buffered soln B,

700bp, 34.3% buffered soln A, 65.7% buffered soln B,

900bp, 33.3% buffered soln A, 66.7% buffered soln B, wherein,

Buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml;

Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml;

Flow velocity: 0.9mL/min;

Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;

Applied sample amount: PCR product 10 μ L.

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