CN102839218B - Method for detecting trichina and cysticercus cellulosae in food - Google Patents
Method for detecting trichina and cysticercus cellulosae in food Download PDFInfo
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- CN102839218B CN102839218B CN2012103496847A CN201210349684A CN102839218B CN 102839218 B CN102839218 B CN 102839218B CN 2012103496847 A CN2012103496847 A CN 2012103496847A CN 201210349684 A CN201210349684 A CN 201210349684A CN 102839218 B CN102839218 B CN 102839218B
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Abstract
The invention provides a method for detecting trichina and cysticercus cellulosae in a food, relates to a method for detecting the trichina and the cysticercus cellulosae, and solves the problem that the existing method for diagnosing food-borne parasites can only detect one of the trichina and the cysticercus cellulosae. The method comprises the following steps of: 1, designing special primers of the trichina and the cysticercus cellulosae and utilizing biotin to label; 2, treating a sample; 3, carrying out double-PCR (Polymerase Chain Reaction) amplification; 4, decorating a probe; 5, coupling a micro-balloon with a light spectrum code of 38 and a decorated trichina Ts gene; coupling a micro-balloon with a light spectrum code of 44 and a cysticercus cellulosae Cc gene; 6, preparing a micro-balloon working solution; 7, preparing a color developing solution; and 8, placing a reaction tube and a negative contrast tube into a PCR instrument; and carrying out cross linking and reading to finish the detection of the trichina and the cysticercus cellulosae in the food. The method disclosed by the invention is used for detecting the trichina and the cysticercus cellulosae.
Description
Technical field
The present invention relates to a kind of method that detects Trichinella spiralis and cysticercus cellulosae.
Background technology
Trichonematosis (Trichinosis) is parasitized the domestic animals such as pig and causes by trichina(Trichinella spiralis) (Trichinella spiralis), except pig, nearly 150 kinds of animals such as dog, cat, mouse, rabbit, ox, sheep, people all can infect, due to this sick infection genesis in having ingested that give birth to or underdone pork or other animal meat containing trichnsuno eyst, and the people infects trichonematosis can cause death, a kind of very serious Zoonosis parasitosis, so be essential items for inspection in the meat hygiene check.Except Australia and some island, its almost extend over the entire globe various places that distribute, become a kind of global disease of natural focus at present.Intestines Trichinella spiralis and flesh Trichinella spiralis (being adult and larva) parasitize in same host, and the stage of mainly causing a disease is muscle larvae and very large to harm, and severe infections often can the causing death.Clinical symptom be take heating, muscular soreness of whole body as main, and the person of being in a bad way can be dead because of toxicaemia, myocarditis and respiratory tract complication.
Cysticercosis cellulosae (Cysticercosis cellulosae), that larva cysticercus cellulosae (Cysticercus cellulosae) by taeniasis suis parasitizes in the animal bodies such as people and pig and a kind of tapeworm larva disease caused is a kind of very important Zoonosis parasitosis.Be one of big event of meat hygiene check, because the pork containing cysticercus cellulosae can not eat, to livestock industry, cause huge financial loss.The cysticercosis cellulosae Major Epidemic is in some countries and regions such as inferior, non-, as to draw, and it is divided into three kinds of musculus cutaneus type, brain type and ocular forms cysticercosis, wherein with the sickness rate of brain type cysticercosis for the highest.
The parasitic method of current diagnosis food source property, although easy and simple to handle, mostly, for once only detecting a kind of polypide, limited to a large extent the parasitic rapid detection of food source property and identification capacity.
Summary of the invention
The present invention will solve the problem that the parasitic method of current diagnosis food source property can only detect a kind of polypide, and a kind of method that detects Trichinella spiralis and cysticercus cellulosae in food is provided.
The present invention detects the method for Trichinella spiralis and cysticercus cellulosae in food, carries out according to the following steps:
One, design Trichinella spiralis Ts gene-specific primer and cysticercus cellulosae Cc Auele Specific Primer, and every pair of primer upstream 5 ' end is all adopted to biotin labeling, primer sequence is as follows:
Trichinella spiralis Ts gene-specific primer:
TsP 1：5’-Biotin-AGGTTCGAAGGCGATCAGATAC-3’，
TsP2：5’-CTGCTCGCCGAGTCTTAAATG-3’，
Cysticercus cellulosae Cc Auele Specific Primer:
CcP3：5’-3iotin-AGCGACGAGACGGGCATAC-3’，
CcP4：5’-CGCTGCTGTTGACTGATGATG-3’
Two, sample preparation: extract the genomic dna of sample to be checked with the DNA extraction test kit, as the pcr amplification template;
Three, double PCR amplification: the genomic dna that the step 1 of take obtains is template, with primer TsP1, TsP2, CcP3 and the CcP4 of step 1, carries out pcr amplification, obtains pcr amplification product;
Four, the modification of probe: design Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe add respectively the connecting arm of 11 T, and carry out the amination modification at 5 of Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe ' end;
Five, the Trichinella spiralis Ts gene probe after spectrum being numbered to 38 microballoon and modifying is coupled, and dilution obtains the microballoon liquid A that concentration is 45000～50000/μ L, cysticercus cellulosae Cc gene probe after spectrum is numbered to 44 microballoon and modifies is coupled, and dilution obtains the microballoon liquid B that concentration is 45000～50000/μ L;
Six, preparation microballoon working fluid: add successively 1 μ L microballoon liquid A and 1 μ L microballoon liquid B in 300 μ L 1.5 * TMAC hybridization solutions, mix, be the microballoon working fluid;
Seven, preparation nitrite ion: mix with SA-PE with 1 * TMAC hybridization solution, be mixed with the nitrite ion of 10 μ g/mL;
Eight, reaction tubes and negative control pipe are set, add successively the microballoon working fluid of 20 μ L, pcr amplification product and the 7 μ L 1 * TE Buffer that 3 μ L step 2 obtain in reaction tubes, microballoon working fluid and 10 μ L 1 * TE Buffer to adding 20 μ L in the negative control pipe, mix with liquid-transfering gun; Then reaction tubes and negative control pipe are placed in to the PCR instrument, through 95 ℃ of sex change 5min, again through 50 ℃ of hybridization 20min, then add 80 μ L nitrite ions in every pipe, mix, reaction tubes and negative control pipe are placed in to the PCR instrument again, 50 ℃ of hybridization 20min, the copper coin of pipe being put into to Luminex100 liquid suspension dot matrix detector after hybridization finishes carries out reading, completes the detection of Trichinella spiralis and cysticercus cellulosae in food; Wherein in step 4, Trichinella spiralis Ts gene probe Tsp is 5 '-CCAACCAGCGATTCGCCGAAGT-3 ', and cysticercus cellulosae Cc gene probe Ccp is 5 '-CACGAGCTTGAGGCAGACTAGGCACC-3 '.
Beneficial effect of the present invention comprises:
(1) the inventive method can detect two kinds of food source property parasites (Trichinella spiralis and cysticercus cellulosae) simultaneously, once can complete the detection of Trichinella spiralis and two kinds of polypides of cysticercus cellulosae, has improved detection efficiency.
(2) PCR system of the present invention is double PCR, and 2 DNA fragmentations can increase simultaneously.The proportioning of various primers in the Design of length of the DNA fragmentation that designs, increases by primer sequence, primer system, realized the equilibrium amplification of 2 gene fragments.
(3) in primer system of the present invention, select crucial upstream primer to carry out mark, each DNA fragmentation that amplification obtains, with biotin labeling, conveniently detects.
(4) the present invention adopts the Luminex technology, is coupled to the probe of fluorescent microsphere and gene fragment that pcr amplification obtains and hybridizes in liquid phase environment, and than the liquid-solid phase hybridization of traditional gene chip, hybridization efficiency is higher, better effects if.In addition, crossover process can be carried out on common 96 orifice plates, can use the hyperchannel volley of rifle fire to carry out pipetting, and therefore whole crossover process is convenient fast, can effectively realize high throughput testing.
The accompanying drawing explanation
The sensitivity test detected result that Fig. 1 is Trichinella spiralis in embodiment five; The sensitivity test detected result that Fig. 2 is cysticercus cellulosae in embodiment five.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the method for Trichinella spiralis and cysticercus cellulosae in present embodiment detection food, carry out according to the following steps:
One, design Trichinella spiralis Ts gene-specific primer and cysticercus cellulosae Cc Auele Specific Primer, and every pair of primer upstream 5 ' end is all adopted to biotin labeling, primer sequence is as follows:
Trichinella spiralis Ts gene-specific primer:
TsP1：5’-Biotin-AGGTTCGAAGGCGATCAGATAC-3’，
TsP2：5’-CTGCTCGCCGAGTCTTAAATG-3’，
Cysticercus cellulosae Cc Auele Specific Primer:
CcP3：5’-Biotin-AGCGACGAGACGGGCATAC-3’，
CcP4：5’-CGCTGCTGTTGACTGATGATG-3’
Two, sample preparation: extract the genomic dna of sample to be checked with the DNA extraction test kit, as the pcr amplification template;
Three, double PCR amplification: the genomic dna that the step 1 of take obtains is template, with primer TsP1, TsP2, CcP3 and the CcP4 of step 1, carries out pcr amplification, obtains pcr amplification product;
Four, the modification of probe: design Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe add respectively the connecting arm of 11 T, and carry out the amination modification at 5 of Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe ' end;
Five, the Trichinella spiralis Ts gene probe after spectrum being numbered to 38 microballoon and modifying is coupled, and dilution obtains the microballoon liquid A that concentration is 45000～50000/μ L, cysticercus cellulosae Cc gene probe after spectrum is numbered to 44 microballoon and modifies is coupled, and dilution obtains the microballoon liquid B that concentration is 45000～50000/μ L;
Six, preparation microballoon working fluid: add successively 1 μ L microballoon liquid A and 1 μ L microballoon liquid B in 300 μ L 1.5 * TMAC hybridization solutions, mix, be the microballoon working fluid;
Seven, preparation nitrite ion: mix with SA-PE with 1 * TMAC hybridization solution, be mixed with the nitrite ion of 10 μ g/mL;
Eight, reaction tubes and negative control pipe are set, add successively the microballoon working fluid of 20 μ L, pcr amplification product and the 7 μ L 1 * TE Buffer that 3 μ L step 2 obtain in reaction tubes, microballoon working fluid and 10 μ L 1 * TE Buffer to adding 20 μ L in the negative control pipe, mix with liquid-transfering gun; Then reaction tubes and negative control pipe are placed in to the PCR instrument, through 95 ℃ of sex change 5min, again through 50 ℃ of hybridization 20min, then add 80 μ L nitrite ions in every pipe, mix, reaction tubes and negative control pipe are placed in to the PCR instrument again, 50 ℃ of hybridization 20min, the copper coin of pipe being put into to Luminex100 liquid suspension dot matrix detector after hybridization finishes carries out reading, completes the detection of Trichinella spiralis and cysticercus cellulosae in food; Wherein in step 4, Trichinella spiralis Ts gene probe Tsp is 5 '-CCAACCAGCGATTCGCCGAAGT-3 ', and cysticercus cellulosae Cc gene probe Ccp is 5 '-CACGAGCTTGAGGCAGACTAGGCACC-3 '.
Be provided with two laser probes in the sense channel of Luminex100 liquid suspension dot matrix detector, a probe can be launched the laser of 635nm wavelength, but two kinds of fluoresceins of excitation labeling microballoon, thus identify different colour code microballoons, carry out qualitative detection; Another laser probe can excite the fluorescein of reporter molecules, by measure microballoon with the intensity of reporter molecules fluorescent signal, can carry out detection by quantitative.
In the result of present embodiment Luminex100 liquid suspension dot matrix detector reading, if spectrum is numbered 3 times of the fluorescence intensity level of 38 microballoon>100 and negative control value, illustrate that sample to be checked contains Trichinella spiralis, if spectrum is numbered 3 times of the fluorescence intensity level of 44 microballoon>100 and negative control value, illustrate that sample to be checked contains cysticercus cellulosae, if the fluorescence intensity level of the microballoon of two kinds of spectrum numbering all>100 and 3 times of negative control value, illustrates that sample to be checked not only contains Trichinella spiralis but also contain cysticercus cellulosae.
Present embodiment the primer and probe are given birth to the biological company limited of work by Shanghai and are synthesized.
In step 1, the DNA extraction test kit is for buying from day TIANamp Genomic DNAKit of root biochemical corp.
SA-PE (SA-PE), spectrum are numbered 38 microballoon and spectrum and are numbered 44 microballoon purchased from the saturating scape bio tech ltd in Shanghai; EDC (carbodiimide), be coupled solvent MES (fatty alcohol-polyoxyethylene ether (3) sulfo group tiger amber acid monoester disodium), TMAC (tetramethylammonium chloride) purchased from Sigma company.
1 * TMAC hybridization solution (50mL) is comprised of the EDTA (pH8.0) of Tris-HCl (pH8.0), the 0.4mL 0.5mol/L of Sarkosyl (sodium N-lauroyl sarcosinate), the 2.5mL 1mol/L of the TMAC of 30mL 5mol/L, 0.25mL mass concentration 20% and the distilled water of surplus.1.5 * TMAC hybridization solution (50mL) is comprised of the EDTA (pH8.0) of Tris-HCl (pH8.0), the 0.6mL 0.5mol/L of TMAC, the 0.376mL 20%Sarkosyl of 45mL 5mol/L, 3.75mL 1mol/L and the distilled water of surplus.
In step 8, Luminex100 liquid suspension dot matrix detector is full of nine think of Bioisystech Co., Ltd purchased from Shenzhen.
Embodiment two: present embodiment is different from embodiment one: in step 3, the reaction system of double PCR amplification is 25 μ L, following ingredients, consists of:
| Composition | Consumption |
| 50～100ng/ μ L genomic dna | 1μL |
| 10nmol/ μ L primer TsP1 | 0.8μL |
| 10nmol/ μ L primer TsP2 | 0.8μL |
| 10nmol/ μ L primer CcP3 | 0.5μL |
| 10nmol/ μ L primer CcP4 | 0.5 |
| 10×PCRBuffer | 2.5μL |
| 2.5mmol/L dNTP Mixture | 3μL |
| Ex Taq enzyme | 0.5μL |
| Sterilized water | 15.4μL |
The pcr amplification condition is: 95 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 30 circulations, then 72 ℃ of extension 10min, 4 ℃ of preservations.Other is identical with embodiment one.
Embodiment three: present embodiment is different from embodiment one: the concrete steps that the Trichinella spiralis Ts gene probe after in step 5, spectrum being numbered to 38 microballoon and modifying is coupled are:
Getting concentration is 5.0 * 10
6the spectrum of individual/mL is numbered 38 microballoon, and concussion mixes, the resuspended microballoon of supersound process 20s; Draw 200 μ L microballoons to brown EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; Add the MES (pH 4.5) that 50 μ L concentration are 0.1mol/L in the EP pipe, concussion mixes, and then supersound process 20s adds the Trichinella spiralis Ts gene probe after the modification of 2 μ L 0.1nmol/ μ L, and concussion mixes; Then add the EDC that 2.5 μ L concentration are 10mg/mL in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; Add the EDC that 2.5 μ L concentration are 4mg/mL again in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; To the Tween-20 that adds 1mL volumetric concentration 0.02% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; To the resuspended microballoon of SDS that adds 1mL mass concentration 0.1% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant, adds 1 * TE Buffer (pH8.0) 100mL, and concussion mixes resuspended microballoon; Being placed in 4 ℃ keeps in Dark Place.Other is identical with embodiment one.
Embodiment four: present embodiment is different from embodiment one: the concrete steps that the cysticercus cellulosae Cc gene probe after in step 5, spectrum being numbered to 44 microballoon and modifying is coupled are:
Getting concentration is 5.0 * 10
6the spectrum of individual/mL is numbered 44 microballoon, and concussion mixes, the resuspended microballoon of supersound process 20s; Draw 200 μ L microballoons to brown EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; Add the MES (pH 4.5) that 50 μ L concentration are 0.1mol/L in the EP pipe, concussion mixes, and then supersound process 20s adds the cysticercus cellulosae Cc gene probe after the modification of 2 μ L 0.1nmol/ μ L, and concussion mixes; Then add the EDC (carbodiimide) that 2.5 μ L concentration are 10mg/mL in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; Add the EDC that 2.5 μ L concentration are 4mg/mL again in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; To the Tween-20 that adds 1mL volumetric concentration 0.02% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; To the resuspended microballoon of SDS that adds 1mL mass concentration 0.1% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant, adds 1 * TE Buffr (pH8.0) 100mL, and concussion mixes resuspended microballoon; Being placed in 4 ℃ keeps in Dark Place.Other is identical with embodiment one.
Embodiment five: the method for Trichinella spiralis and cysticercus cellulosae in present embodiment detection food, carry out according to the following steps:
One, the Trichinella spiralis 18S rDNA logined according to GenBank (accession number: AY497012) with cysticercus cellulosae ITS1 (accession number: gene order EU747668), utilize Primer 5.0 and Oligo 6.0 design Trichinella spiralis Ts gene-specific primer and cysticercus cellulosae Cc Auele Specific Primers, and every pair of primer upstream 5 ' end is all adopted to biotin labeling, primer sequence is as follows:
Trichinella spiralis Ts gene-specific primer:
TsP 1：5’-Biotin-AGGTTCGAAGGCGATCAGATAC-3’，
TsP2：5’-CTGCTCGCCGAGTCTTAAATG-3’，
Cysticercus cellulosae Cc Auele Specific Primer:
CcP3：5’-Biotin-AGCGACGAGACGGGCATAC-3’，
CcP4：5’-CGCTGCTGTTGACTGATGATG-3’
Two, sample preparation: extract the genomic dna of sample to be checked with DNA extraction test kit (buying from day TIANamp Genomic DNA Kit of root biochemical corp), as the pcr amplification template;
Three, double PCR amplification: the genomic dna that the step 1 of take obtains is template, with primer TsP1, TsP2, CcP3 and the CcP4 of step 1, carries out pcr amplification, obtains pcr amplification product;
The reaction system of double PCR amplification is 25 μ L, following ingredients, consists of:
| Composition | Consumption |
| 50～100ng/ μ L genomic dna | 1μL |
| 10nmol/ μ L primer TsP1 | 0.8μL |
| 10nmol/ μ L primer TsP2 | 0.8μL |
| 10nmol/ μ L primer CcP3 | 0.5μL |
| 10nmol/ μ L primer CcP4 | 0.5μL |
| 10×PCRBuffer | 2.5μL |
| 2.5mmol/L dNTP Mixture | 3μL |
| Ex Taq enzyme | 0.5μL |
| Sterilized water | 15.4μL |
The pcr amplification condition is: 95 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 30 circulations, then 72 ℃ of extension 10min, 4 ℃ of preservations.
Four, the modification of probe: design Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe add respectively the connecting arm of 11 T, and carry out the amination modification at 5 of Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe ' end;
Trichinella spiralis Ts gene probe Tsp:5 '-CCAACCAGCGATTCGCCGAAGT-3 '
Cysticercus cellulosae Cc gene probe Ccp:5 '-CACGAGCTTGAGGCAGACTAGGCACC-3 '
Five, the Trichinella spiralis Ts gene probe coupling after spectrum being numbered to 38 microballoon and modifying, and dilution obtains the microballoon liquid A that concentration is 50000/μ L, cysticercus cellulosae Cc gene probe coupling after spectrum is numbered to 44 microballoon and modifies, and dilution obtains the microballoon liquid B that concentration is 50000/μ L;
Six, preparation microballoon working fluid: add successively 1 μ L microballoon liquid A and 1 μ L microballoon liquid B in 300 μ L 1.5 * TMAC hybridization solutions, mix, be the microballoon working fluid;
Seven, preparation nitrite ion: mix with SA-PE with 1 * TMAC hybridization solution, be mixed with the nitrite ion of 10 μ g/mL;
Eight, reaction tubes and negative control pipe are set, add successively the microballoon working fluid of 20 μ L, pcr amplification product and the 7 μ L1 * TE Buffer that 3 μ L step 2 obtain in reaction tubes, microballoon working fluid and 10 μ L 1 * TE Buffer to adding 20 μ L in the negative control pipe, mix with liquid-transfering gun; Then reaction tubes and negative control pipe are placed in to the PCR instrument, through 95 ℃ of sex change 5min, again through 50 ℃ of hybridization 20min, then add 80 μ L nitrite ions in every pipe, mix, reaction tubes and negative control pipe are placed in to the PCR instrument again, 50 ℃ of hybridization 20min, the copper coin of pipe being put into to Luminex100 liquid suspension dot matrix detector after hybridization finishes carries out reading, completes the detection of Trichinella spiralis and cysticercus cellulosae in food.
The present embodiment method is carried out to following checking:
(1) checking of probe coupling:
With with probe Tsp and Ccp reverse complemental and through biotin labeled oligonucleotide, replacing the PCR product to test positive control as this, utilize nucleic acid hybridization, add each reverse complementary sequence successively in mixing microballoon, whether correctly be linked on special microballoon in order to verify probe.
The operation steps of probe coupling checking is:
1. take out the microballoon of coupling probe from 4 ℃, concussion is outstanding shakes, supersound process 20s;
2. prepare one group and mix the microballoon working fluid: with 1.5 * TMAC hybridization solution, the microballoon of each group coupling probe is diluted to 150/μ L;
Concussion outstanding shake, supersound process microballoon 20s;
4. get 96 orifice plates, 4 positive reactions and a blank are set, to each positive and blank hole, respectively add 20 μ L mixing microballoon working fluids;
5. add 10 μ L 1 * TE Buffer (pH8.0) in each blank well, and add 3 μ L antitone sequences and 7 μ L 1 * TE Buffer (pH8.0) to each positive control hole;
With the upper and lower pressure-vaccum of liquid-transfering gun for several times to mix each reacting hole;
7. build Sptting plate, be placed in the PCR instrument, 95 ℃ of sex change 5min, then through 50 ℃ of hybridization 20min;
8. prepare fresh nitrite ion: with 1 * TMAC hybridization solution, SAPE is made into to 10 μ g/mL (every hole needs 25 μ L nitrite ions);
9. every hole adds 25 μ L nitrite ions, and with the upper and lower pressure-vaccum of liquid-transfering gun for several times to mix each reacting hole;
10. standing 5min under 50 ℃ of hybridization temperatures, keep fixing temperature, draws the detection of 50 μ L/ pipes for Luminex100 liquid suspension dot matrix detector, according to the crosslinked situation of detected result judgement probe and microballoon.
The result that probe is coupled checking is: the sample of having hybridized is placed on the Luminex100 chip instrument, before use, need instrument preheating 30min, drawing 50 μ L/ pipes is detected, do 3 parallel detections for every group and get its mean value, it is as shown in table 1 that probe is coupled result, and from numerical value, every kind of probe antitone sequence all has good combination with corresponding probe while reacting with mixing microballoon working fluid, and fluorescent value is very high, and extremely low with the combined with fluorescent value of non-own probe.3 times of fluorescence intensity level>100 and negative control values are effective detected value.Result shows that probe can correctly be linked on special microballoon, and it is higher to be coupled efficiency.
Table 1 probe is coupled result
(2) specific test:
Respectively with the primer of Trichinella spiralis and cysticercus cellulosae by the condition amplification Trichinella spiralis after optimizing and the mixed genomic DNA of cysticercus cellulosae, then apply the fluorescence intensity that liquid phase gene chip system detects each PCR product, the TE solution of usining replaces the PCR product as negative control, using reverse complementary sequence as positive control, add during detection and mix the microballoon working fluid in order to verify the specificity of present method.
The specific test result: fluorescence intensity level is effective detected value much larger than 3 times of negative control values, and specificity is good, in Table 2.
Table 2 specific test result
| Detect target | The Trichinella spiralis fluorescence intensity | The cysticercus cellulosae fluorescence intensity |
| Negative control | 36 | 41 |
| Positive control | 4592 | 1854 |
| Trichinella spiralis primer amplified product | 3309.5 | 8.5 |
| Cysticercus cellulosae primer amplified product | 6.5 | 870 |
(3) sensitivity test:
The learnt from else's experience PCR product of gradient dilution, detected by the present embodiment method, with TE solution, replaces the negative contrast of PCR product, with numeral 0～12, represents successively 2
0(being PCR product stoste)～2
-12weaker concn.
The sensitivity test result: as shown in Figure 1, result shows that Trichinella spiralis negative control fluorescence intensity is 34 to the sensitivity test detected result of Trichinella spiralis, utilizes the present embodiment method to reach 2 of stoste to the minimum detectable concentration of Trichinella spiralis
-9, about 53.52ng/mL, fluorescence intensity 174 is about 4.8 times of negative control.
As shown in Figure 2, cysticercus cellulosae negative control fluorescence intensity is 63 to the sensitivity test detected result of cysticercus cellulosae, utilizes the present embodiment method to reach 2 of stoste to the minimum detectable concentration of cysticercus cellulosae
-8, about 87.89ng/mL, fluorescence intensity 331 is about 5.1 times of negative control.
(4) the double PCR product detects
Get the hybrid dna of two kinds of polypides, carry out double pcr amplification and liquid phase genechip detection, detected result is in Table 3, and the type of the PCR product that in table, data presentation adds is consistent with the positive findings of hybridization signal gained, and the hybridization phenomenon does not cause confusion.
Table 3 double PCR product detected result
| Detect target | The Trichinella spiralis fluorescence intensity | The cysticercus cellulosae fluorescence intensity |
| Negative control | 23 | 15 |
| Hybrid dna double PCR amplified production | 3546 | 972.5 |
(5) manual simulation and clinical sample detect test
Extract the genomic dna through the pork for Artificial simulative experiments of quarantine; Extract 3 parts of pork clinical sample genomic dnas of censorship.To divide for the genomic dna of Artificial simulative experiments pork and install to 20 EP pipes, every pipe 20 μ L, and add at random Trichinella spiralis and cysticercus cellulosae genomic dna, and the genomic dna of submitted sample is packed as 20 μ L/ pipes.Detect manual simulation and clinical totally 23 parts of sample DNAs with the mix primer amplifying genom DNA, finally by the present embodiment method, detect the PCR product, and result is analyzed.
The manual simulation detects test-results with clinical sample: the Blind Test result conforms to substantially with expected result, and 3 parts of meat sample detected results of censorship are all negative.Fluorescence intensity level and negative value are basicly stable, show that the method has feasibility in actually operating.
Claims (3)
1. a method that detects Trichinella spiralis and cysticercus cellulosae in food, is characterized in that detecting the method for Trichinella spiralis and cysticercus cellulosae in food, carries out according to the following steps:
One, design Trichinella spiralis Ts gene-specific primer and cysticercus cellulosae Cc Auele Specific Primer, and every pair of primer upstream 5 ' end is all adopted to biotin labeling, primer sequence is as follows:
Trichinella spiralis Ts gene-specific primer:
TsP1：5’-Biotin-AGGTTCGAAGGCGATCAGATAC-3’，
TsP2：5’-CTGCTCGCCGAGTCTTAAATG-3’，
Cysticercus cellulosae Cc Auele Specific Primer:
CcP3：5’-Biotin-AGCGACGAGACGGGCATAC-3’，
CcP4：5’-CGCTGCTGTTGACTGATGATG-3’
Two, sample preparation: extract the genomic dna of sample to be checked with the DNA extraction test kit, as the pcr amplification template;
Three, double PCR amplification: the genomic dna that the step 2 of take obtains is template, with primer TsP1, TsP2, CcP3 and the CcP4 of step 1, carries out pcr amplification, obtains pcr amplification product;
Four, the modification of probe: design Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe add respectively the connecting arm of 11 T, and carry out the amination modification at 5 of Trichinella spiralis Ts gene probe and cysticercus cellulosae Cc gene probe ' end;
Five, the Trichinella spiralis Ts gene probe after spectrum being numbered to 38 microballoon and modifying is coupled, and dilution obtains the microballoon liquid A that concentration is 45000～50000/μ L, cysticercus cellulosae Cc gene probe after spectrum is numbered to 44 microballoon and modifies is coupled, and dilution obtains the microballoon liquid B that concentration is 45000～50000/μ L;
Six, preparation microballoon working fluid: add successively 1 μ L microballoon liquid A and 1 μ L microballoon liquid B in 300 μ L1.5 * TMAC hybridization solution, mix, be the microballoon working fluid;
Seven, preparation nitrite ion: mix with SA-PE with 1 * TMAC hybridization solution, be mixed with the nitrite ion of 10 μ g/mL;
Eight, reaction tubes and negative control pipe are set, add successively the microballoon working fluid of 20 μ L, pcr amplification product and the 7 μ L1 * TE Buffer that 3 μ L step 2 obtain in reaction tubes, microballoon working fluid and 10 μ L1 * TE Buffer to adding 20 μ L in the negative control pipe, mix with liquid-transfering gun; Then reaction tubes and negative control pipe are placed in to the PCR instrument, through 95 ℃ of sex change 5min, again through 50 ℃ of hybridization 20min, then add 80 μ L nitrite ions in every pipe, mix, reaction tubes and negative control pipe are placed in to the PCR instrument again, 50 ℃ of hybridization 20min, the copper coin of pipe being put into to Luminex100 liquid suspension dot matrix detector after hybridization finishes carries out reading, completes the detection of Trichinella spiralis and cysticercus cellulosae in food; Wherein in step 4, Trichinella spiralis Ts gene probe Tsp is 5 '-CCAACCAGCGATTCGCCGAAGT-3 ', and cysticercus cellulosae Cc gene probe Ccp is 5 '-CACGAGCTTGAGGCAGACTAGGCACC-3 ';
Wherein, in step 3, the reaction system of double PCR amplification is 25 μ L, following ingredients, consists of:
The pcr amplification condition is: 95 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 30 circulations, then 72 ℃ of extension 10min, 4 ℃ of preservations.
2. a kind of method that detects Trichinella spiralis and cysticercus cellulosae in food according to claim 1, the concrete steps that the Trichinella spiralis Ts gene probe after it is characterized in that in step 5 spectrum is numbered 38 microballoon and modifying is coupled are:
Getting concentration is 5.0 * 10
6the spectrum of individual/mL is numbered 38 microballoon, and concussion mixes, the resuspended microballoon of supersound process 20s; Draw 200 μ L microballoons to brown EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; Add the MES that 50 μ L concentration are 0.1mol/L in the EP pipe, concussion mixes, and then supersound process 20s adds the Trichinella spiralis Ts gene probe after the modification of 2 μ L0.1nmol/ μ L, and concussion mixes; Then add the EDC that 2.5 μ L concentration are 10mg/mL in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; Add the EDC that 2.5 μ L concentration are 4mg/mL again in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; To the Tween-20 that adds 1mL volumetric concentration 0.02% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; To the resuspended microballoon of SDS that adds 1mL mass concentration 0.1% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant, adds 1 * TE Buffer100mL, and concussion mixes resuspended microballoon; Being placed in 4 ℃ keeps in Dark Place.
3. a kind of method that detects Trichinella spiralis and cysticercus cellulosae in food according to claim 1, the concrete steps that the cysticercus cellulosae Cc gene probe after it is characterized in that in step 5 spectrum is numbered 44 microballoon and modifying is coupled are:
Getting concentration is 5.0 * 10
6the spectrum of individual/mL is numbered 44 microballoon, and concussion mixes, the resuspended microballoon of supersound process 20s; Draw 200 μ L microballoons to brown EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; Add the MES that 50 μ L concentration are 0.1mol/L in the EP pipe, concussion mixes, and then supersound process 20s adds the cysticercus cellulosae Cc gene probe after the modification of 2 μ L0.1nmol/ μ L, and concussion mixes; Then add the EDC that 2.5 μ L concentration are 10mg/mL in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; Add the EDC that 2.5 μ L concentration are 4mg/mL again in the EP pipe, concussion mixes rear lucifuge, room temperature is placed 30min; To the Tween-20 that adds 1mL volumetric concentration 0.02% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant; To the resuspended microballoon of SDS that adds 1mL mass concentration 0.1% in the EP pipe, the centrifugal 2min of 8000r/min, abandon supernatant, adds 1 * TE Buffer100mL, and concussion mixes resuspended microballoon; Being placed in 4 ℃ keeps in Dark Place.
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