CN104789698A - Triple RT-PCR detection kit - Google Patents
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Abstract
The invention belongs to the field of biological detection and in particular relates to a triple RT-PCR detection kit. Three pairs of specific primers are respectively designed according to PEDV M gene, TGEV N gene and GAR VP 7 gene, wherein the sizes of the amplified fragments are 199bp, 499bp and 333bp respectively; a user can directly judge by naked eyes according to electrophoretic results. The kit is high in specificity, high in repeatability and convenient and quick to operate; single infection or mixed infection can be definitely diagnosed in 4-6 hours generally; the double purposes of diagnosing and identifying can be achieved. The kit is higher in sensitivity; the minimum detection concentration of porcine epidemic diarrhea virus is 4.3 ng per microliter; the minimum detection concentration of porcine transmissible gastroenteritis virus is 3.2 ng per microliter; the minimum detection concentration of rotavirus diarrhea of porcine group A is 6.1 ng per microliter.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of triple RT-PCR detection kit.
Background technology
The cardinal symptom that transmissible gastro-enteritis virus (PEDV) infects is vomiting, watery diarrhea, weight loss and serious dehydration with serious stench.5 week age, above pig mortality ratio was lower, and on Adult Pig, morbidity is gentle, and may occur poor appetite, diarrhoea, milking sow may occur agalasisa.But the most easy infection of the piglet below 2 week age, and incidence and mortality is higher, may by hunger, dehydration and oxypathy cause death.This test kit is applicable to the auxiliary diagnosis to Porcine epidemic diarrhea virus.
The cardinal symptom that Porcine epidemic diarrhea virus (TGEV) infects is watery diarrhea, or vomiting after eating; Sick temperature of pig body is normal or slightly high, and spirit is depressed, appetite stimulator or absolutely useless; Wean pig, sow are often about a week of down in spirits, apocleisis and persistent diarrhea, and recover normal gradually, retarded growth after minority pig recovers; Fattening pig is all suffered from diarrhoea after raising infection with circle, rehabilitation after a week, mortality ratio 1%--3%.
The cardinal symptom that pig A rotavirus (GAR) infects is acute diarrhea, and piglet is multiple, and main manifestations is spirit tired, apocleisis, vomiting, diarrhoea, dewater, the clinical symptom such as lose weight.Adult Pig and bred pigs mostly are inapparent infection, be cause the common disease of pig gastro-enteritis because of.This disease finds in the children suffering from diarrhoea early than nineteen forty-three, within 1975, from pig, isolate rotavirus first, this disease is in worldwide popular at present, rotavirus illness is a kind of infectious diseases common to human beings and animals, the mankind and many livestock and poultry can be encroached on, not only infection rate is high, and sickness rate is also quite high, endangers larger to human health and livestock industry.
At present, the common method for detecting PEDV, TGEV and GAR has the detection of virus purification, fluorescence antibody, immunohistochemical method, Electronic Speculum, ELISA Pathogen test method and RT-PCR etc.But virus purification takes time and effort and more difficult; Fluorescence antibody detect and immunohistochemical method non-specific higher and need fluorescent microscope to observe; ELISA the Methods of Detection of Pathogens Application comparison is extensive, but it is comparatively complicated to compare PCR method process; Conventional RT-PCR can only detect a kind of cause of disease at every turn and need again to carry out PCR, complex operation by after RNA reverse transcription, easily causes Aerosol Pollution.
Therefore the present invention designs the triple RT-PCR detection kit of a kind of single stage method, and simplify the operation step, only just can determine Simple infection or polyinfection by One_step PCR, and can make a definite diagnosis infection viral species.This test kit has the advantages such as easy and simple to handle, accurate, quick.
Summary of the invention
The object of this invention is to provide a kind of triple RT-PCR detection kit, detection can be made fast and accurately.
The present invention for the taked technical scheme that solves the problem is: a kind of triple RT-PCR detection kit, this test kit is formed by the A box extracted for viral RNA with for the B box that RT-PCR detects, damping fluid RG 9 mL is provided with in A box, damping fluid RF 3 mL, damping fluid RD 8 mL, damping fluid RW 12 mL, RNA elutriant 3 mL, 50 times of TAE electrophoretic buffer 8 mL, staining fluid 10 μ L, sample-loading buffer 30 μ L, Rnase-free adsorption column, collection tube 10, centrifuge tube 10 and 0.2 mL PCR pipe 10, amplification reaction solution 220 μ L is provided with in B box, enzyme system 10 μ L, described amplification reaction solution comprises the PCR primer sets for detecting Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig A rotavirus, described primer sets is made up of three groups of primer sequences, and the primer sequence detected for Porcine epidemic diarrhea virus is respectively SEQ ID NO:1 and SEQ ID NO:2, the primer sequence detected for transmissible gastro-enteritis virus is respectively SEQ ID NO:3 and SEQ ID NO:4, the primer sequence detected for pig A rotavirus is respectively SEQ ID NO:5 and SEQ ID NO:6.
The detection method of described triple RT-PCR detection kit, comprises the following steps:
The extraction of step one, template ribonucleic acid
1., in processed sample, add 800 μ L damping fluid RG, after fully putting upside down mixing, at room temperature leave standstill 5min, obtained mixed solution I, for subsequent use;
2. in mixed solution I, add 200 μ L damping fluid RF, at temperature is 30 DEG C, 10min is hatched after mixing, putting into temperature is afterwards 2-8 DEG C, rotating speed is centrifugal 15min in the whizzer of 12000rpm, collect supernatant liquor, in supernatant liquor, add 500 μ L damping fluid RD, and hatch 5min at temperature is 15-30 DEG C, obtained mixed solution II, for subsequent use;
3., mixed solution II being proceeded to cover has in the Rnase-free adsorption column of collection tube, after left at room temperature 2min, put into the centrifugal 1min of whizzer that rotating speed is 12000rpm, discard liquid in collection tube, then add the damping fluid RW of 800 μ L in Rnase-free adsorption column, after left at room temperature 2min, putting into the centrifugal 1min of whizzer that rotating speed is 12000rpm, discard liquid in collection tube, is then centrifugal 2min in the whizzer of 12000rpm at rotating speed, to remove residual washings, for subsequent use;
4. the Rnase-free adsorption column after, step 3. being processed puts into centrifuge tube, 50 μ L RNA elutriants are dripped to adsorption film centre, after left at room temperature 2min, put into the centrifugal 1min of whizzer that rotating speed is 12000rpm, collect supernatant liquor and namely obtain template ribonucleic acid;
Step 2, pcr amplification
(1), dosing: take out amplification reaction solution, at room temperature dissolve and mix, mix in the ratio of each reaction amplification reaction solution 21.5 μ L, enzyme system 1 μ L and stir after centrifugal, obtain reaction solution, for subsequent use;
(2), application of sample: reaction solution is dispensed in each PCR reaction tubes by 22.5 μ L/ pipes, in each PCR reaction tubes, adds the one in 2.5 μ L samples, negative controls or positive control solution respectively, close after be transferred to nucleic acid amplification district, for subsequent use;
(3), pcr amplification detects: the PCR reaction tubes that step (2) processes is put into PCR instrument and increased, obtained amplified production, amplification condition is: heat up 48 DEG C of 10min, denaturation 94 DEG C of 2min, sex change 94 DEG C of 10s, anneal 57 DEG C of 30s, extends 72 DEG C of 20s, 30 circulations, draw solubility curve 72 DEG C of 5min;
Step 3, electrophoresis
The TAE electrophoretic buffer of agarose and 50 times of dilutions is added in container, and 50 times of TAE electrophoretic buffer 66.7mL diluted that every gram of agarose is corresponding, the container that agarose and 50 times of TAE electrophoretic buffers are housed is put into microwave oven dissolve, then in container, add 5 μ L staining fluids, mix and obtained sepharose liquid after stirring, amplified production is placed in the TAE electrophoretic buffer electrophoresis of 110 ~ 120V voltage, 50 times of dilutions, observations under ultraviolet lamp;
Step 4, result judge
If the track of negative control, blank is without band or the band without 199bp, 499bp, 330bp, and positive control has obvious band at the arbitrary place of 199bp, 499bp or 330bp, then detected result is effective, wherein, test sample occurs that 199bp amplified band is that Porcine epidemic diarrhea virus is positive, occur that 499bp amplified band is that transmissible gastro-enteritis virus is positive, occur that 330bp amplified band is that pig A rotavirus is positive, occur that 2 or 3 amplified bands are then corresponding virus mixed infection, otherwise be negative.
beneficial effect
One, the present invention is according to PEDV
mgene, TGEV
ngene and GAR
vP7 genes design 3 pairs of Auele Specific Primers respectively, and amplified fragments size is 199bp, 499bp and 333bp, and user can directly with the naked eye be judged by electrophoresis result.
Two, test kit high specificity of the present invention, reproducible, convenient to operation, general 4 ~ 6 h just can make a definite diagnosis Simple infection or polyinfection, the dual purpose reaching diagnosis and differentiate.
Three, this test kit has higher susceptibility, and the minimal detectable concentration of Porcine epidemic diarrhea virus is 4.3 ng/ μ L, the minimal detectable concentration of transmissible gastro-enteritis virus is 3.2 ng/ μ L, the minimal detectable concentration of pig A rotavirus is 6.1 ng/ μ L.
Accompanying drawing explanation
fig. 1 is the One step RT-PCR detected result that the present invention carries out PEDV, TGEV and GAR;
Fig. 2 is the specific detection result that the present invention carries out PEDV, TGEV, GAR;
Fig. 3 is the repeatable detected result that the present invention carries out PEDV, TGEV, GAR;
Fig. 4 is the susceptibility detected result that the present invention carries out PEDV;
Fig. 5 is the susceptibility detected result that the present invention carries out TGEV;
Fig. 6 is the susceptibility detected result that the present invention carries out GAR.
Embodiment
A kind of triple RT-PCR detection kit, this test kit is formed by the A box extracted for viral RNA with for the B box that RT-PCR detects, be provided with damping fluid RG 9 mL, damping fluid RF 3 mL, damping fluid RD 8 mL, damping fluid RW 12 mL, RNA elutriant 3 mL, 50 times of TAE electrophoretic buffer 8 mL, staining fluid 10 μ L, sample-loading buffer 30 μ L, Rnase-free adsorption column, collection tube 10, centrifuge tube 10 and 0.2 mL PCR pipe 10 in A box, in B box, be provided with amplification reaction solution 220 μ L, enzyme system 10 μ L; Described amplification reaction solution comprises the PCR primer sets for detecting Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig A rotavirus, described primer sets is made up of three groups of primer sequences, and the primer sequence detected for Porcine epidemic diarrhea virus is respectively SEQ ID NO:1 and SEQ ID NO:2; The primer sequence detected for transmissible gastro-enteritis virus is respectively SEQ ID NO:3 and SEQ ID NO:4; The primer sequence detected for pig A rotavirus is respectively SEQ ID NO:5 and SEQ ID NO:6.
The detection method of described triple RT-PCR detection kit, comprises the following steps:
The extraction of step one, template ribonucleic acid
1., in processed sample, add 800 μ L damping fluid RG, after fully putting upside down mixing, at room temperature leave standstill 5min, obtained mixed solution I, for subsequent use;
2. in mixed solution I, add 200 μ L damping fluid RF, at temperature is 30 DEG C, 10min is hatched after mixing, putting into temperature is afterwards 2-8 DEG C, rotating speed is centrifugal 15min in the whizzer of 12000rpm, collect supernatant liquor, in supernatant liquor, add 500 μ L damping fluid RD, and hatch 5min at temperature is 15-30 DEG C, obtained mixed solution II, for subsequent use;
3., mixed solution II being proceeded to cover has in the Rnase-free adsorption column of collection tube, after left at room temperature 2min, put into the centrifugal 1min of whizzer that rotating speed is 12000rpm, discard liquid in collection tube, then add the damping fluid RW of 800 μ L in Rnase-free adsorption column, after left at room temperature 2min, putting into the centrifugal 1min of whizzer that rotating speed is 12000rpm, discard liquid in collection tube, is then centrifugal 2min in the whizzer of 12000rpm at rotating speed, to remove residual washings, for subsequent use;
4. the Rnase-free adsorption column after, step 3. being processed puts into centrifuge tube, 50 μ L RNA elutriants are dripped to adsorption film centre, after left at room temperature 2min, put into the centrifugal 1min of whizzer that rotating speed is 12000rpm, collect supernatant liquor and namely obtain template ribonucleic acid;
Step 2, pcr amplification
(1), dosing: take out amplification reaction solution, at room temperature dissolve and mix, mix in the ratio of each reaction amplification reaction solution 21.5 μ L, enzyme system 1 μ L and stir after centrifugal, obtain reaction solution, for subsequent use;
(2), application of sample: reaction solution is dispensed in each PCR reaction tubes by 22.5 μ L/ pipes, in each PCR reaction tubes, adds the one in 2.5 μ L samples, negative controls or positive control solution respectively, close after be transferred to nucleic acid amplification district, for subsequent use;
(3), pcr amplification detects: the PCR reaction tubes that step (2) processes is put into PCR instrument and increased, obtained amplified production, amplification condition is: heat up 48 DEG C of 10min, denaturation 94 DEG C of 2min, sex change 94 DEG C of 10s, anneal 57 DEG C of 30s, extends 72 DEG C of 20s, 30 circulations, draw solubility curve 72 DEG C of 5min;
Step 3, electrophoresis
The TAE electrophoretic buffer of agarose and 50 times of dilutions is added in container, and 50 times of TAE electrophoretic buffer 66.7mL diluted that every gram of agarose is corresponding, the container that agarose and 50 times of TAE electrophoretic buffers are housed is put into microwave oven dissolve, then in container, add 5 μ L staining fluids, mix and obtained sepharose liquid after stirring, amplified production is placed in the TAE electrophoretic buffer electrophoresis of 110 ~ 120V voltage, 50 times of dilutions, observations under ultraviolet lamp;
Step 4, result judge
If the track of negative control, blank is without band or the band without 199bp, 499bp, 330bp, and positive control has obvious band at the arbitrary place of 199bp, 499bp or 330bp, then detected result is effective, wherein, test sample occurs that 199bp amplified band is that Porcine epidemic diarrhea virus is positive, occur that 499bp amplified band is that transmissible gastro-enteritis virus is positive, occur that 330bp amplified band is that pig A rotavirus is positive, occur that 2 or 3 amplified bands are then corresponding virus mixed infection, otherwise be negative.
Experiment 1
This Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and the triple RT-PCR detection kit of pig A rotavirus single stage method comprise viral RNA and extract (A box) and One step RT-PCR detection (B box) two parts.A box comprises damping fluid RG 9 mL, damping fluid RF 3 mL, damping fluid RD 8 mL, damping fluid RW 12 mL, RNA elutriant 3 mL, 50 × TAE electrophoretic buffer 8 mL, staining fluid 10 μ L, sample-loading buffer 30 μ L, Rnase-free adsorption column and collection tube 10, centrifuge tube 10,0.2 mL thin-walled PCR pipe 10.B box comprises amplification reaction solution 220 μ L, enzyme system 10 μ L, positive control 20 μ L, negative control 20 μ L.Concrete composition is as follows:
1, damping fluid RG:9mL/ bottle, is sub-packed in Brown Glass Brown glass bottles and jars only, and 4 DEG C save backup.
2, damping fluid RF:3mL/ bottle, is sub-packed in Brown Glass Brown glass bottles and jars only, and 4 DEG C save backup.
3, damping fluid RD:8mL/ bottle, be sub-packed in white plastic bottle, normal temperature saves backup.
4, damping fluid RW:12mL/ bottle, be sub-packed in white plastic bottle, normal temperature saves backup.
5, RNA elutriant: 3mL/ bottle, be sub-packed in white plastic bottle, normal temperature saves backup.
6,50 × TAE electrophoretic buffer: ordinary method is prepared, and 8mL/ bottle, normal temperature saves backup.
7, amplification reaction solution: each reaction comprises One step RT-PCR Buffer12.5 μ L, each 1 μ L of P1, P2, T1, T2, G1, G2, Rnase-free water 3 μ L, totally 21.5 μ L, 10 reactions amount to 215 μ L, and-20 DEG C save backup.Wherein P1, P2, T1, T2, G1, G2 design for the present invention, are given birth to the primer of work synthesis by Shanghai.P1, P2 can increase Porcine epidemic diarrhea virus gene fragment 199bp, and T1, T2 can increase transmissible gastro-enteritis virus gene fragment 499bp, and G1, G2 can increase pig A rotavirus gene fragment 330bp.
8, enzyme system: each reaction 1 μ L, totally 10 μ L ,-20 DEG C save backup.
9, positive control: extract total serum IgE in porcine epizootic diarrhea, transmissible gastroenteritis of swine and pig A rotavirus vaccine, P1, P2, T1, T2, G1, G2 is adopted to carry out RT-PCR respectively, carrier T is connected after being reclaimed by object fragment glue, transform DH5 α, extract plasmid and be diluted to suitable concn as positive control, each reaction 2.5 μ L, totally 25 μ L/ manage.
10, negative control: each reaction 2.5 μ L, totally 25 μ L/ manage.
11, staining fluid: 10 μ L/ manage, is sub-packed in normal temperature in brown centrifuge tube and preserves.
12, sample-loading buffer: 30 μ L/ manage, is sub-packed in normal temperature in brown centrifuge tube and preserves.
The present invention also provides a kind of mentioned reagent box that utilizes to carry out the detection method of One step RT-PCR to porcine epizootic diarrhea, transmissible gastroenteritis of swine and pig A rotavirus, and concrete operation step is as follows:
One, sample preparation:
1, sample collecting: the pig dying of illness or slaughter, gets the tissue such as stomach, intestines or the movement such as intestinal contents, ight soil; Live hog to be checked, gets blood 5mL with syringe.2 ~ 8 DEG C of preservations, send test in laboratory.(require that censorship pathological material of disease is fresh, forbid multigelation.）
2, sample preparation: every increment product process respectively
2.1 tissue sample process: every part of tissue at random takes sample from three different positions and is about 1g, get 0.05g after shredding mixing with operating scissors to grind in mill, add 1.5mL physiological saline and continue grinding, go in 1.5mL sterile centrifugation tube after homogenate, the centrifugal 2min of 8000rpm, gets supernatant liquor 100 μ L in 1.5mL sterile centrifugation tube.
2.2, whole blood sample process: get serum 100 μ L after blood clotting, put in 1.5mL sterile centrifugation tube.
Two, operation steps
1. nucleic acid extraction (A box):
1.1 get processed sample, and add 800 μ L damping fluid RG respectively, fully put upside down mixing, room temperature leaves standstill 5min.
1.2 add 200 μ L damping fluid RF, firmly rock 15s mixing, hatch 10min, the centrifugal 15min of 12000rpm at 2-8 DEG C at 30 DEG C.
Supernatant is centrifugally carefully drawn (trying not during absorption to be drawn onto suspended impurity) and proceeds in new centrifuge tube by 1.3 afterwards, adds 500 μ L damping fluid RD, hatches 5min at 15-30 DEG C.
Liquid in centrifuge tube is added (adsorption column will put collection tube, and adsorption column volume is 800 μ L, can add if sample volume is greater than 800 μ L in batches) in adsorption column by 1.4, and room temperature leaves standstill 2min, the centrifugal 1min of 12000rpm.
1.5 discard liquid in collection tube, and in adsorptivity, add the damping fluid RW of 800 μ L, room temperature leaves standstill 2min, the centrifugal 1min of 12000rpm.Discard liquid in collection tube, the centrifugal 2min of 12000rpm, to remove residual washings.
Adsorption column to proceed in a new centrifuge tube by 1.6, and to the unsettled dropping 50 μ L RNA elutriant of adsorption film central authorities, room temperature leaves standstill 2min, and the centrifugal 1min of 12000rpm, in centrifuge tube, liquid is template ribonucleic acid.
2, pcr amplification (B box)
2.1 dosings (carrying out in reagent area in preparation): take out amplification reaction solution, room temperature is fully mixing after melting, of short duration centrifugal, and to extract reaction solution with enzyme according to following proportioning preparation reaction solution respectively by reaction number: (noting considering having positive control and negative control even blank)
Should fully mix during reaction solution preparation, of short duration centrifugal, be dispensed in PCR reaction tubes by 22.5 μ L/ pipes, be transferred to sample process district;
2.2, application of sample (carrying out in sample process district): add sample, negative control, positive control that 2.5 μ L handle well respectively, build pipe lid, be transferred to nucleic acid amplification district;
2.3, pcr amplification detects (carrying out in nucleic acid amplification district): reaction tubes is placed in PCR instrument sample cell, arranging amplification reaction condition is: 48 DEG C of 10min, 94 DEG C of 2min; 94 DEG C of 10s, 57 DEG C of 30s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 5min.
3. electrophoresis
Claim 1.5g agarose to be put in 500mL Erlenmeyer flask, the TAE electrophoretic buffer 100mL(adding 50 times of dilutions gets 2mL 50 times of TAE electrophoretic buffers, is diluted to 100mL with bi-distilled water), melt in microwave oven, add 5 μ L staining fluid mixings.In electrophoresis chamber, put comb well, pour sepharose into, after to be solidified by pcr amplification product 5 μ L point sample in sepharose hole, with 110 ~ 120V voltage in 50 times dilution TAE electrophoretic buffer in electrophoresis, observations under ultraviolet lamp.
Three, result judges:
The track of negative control, blank is without band or the band without 199bp, 499bp, 330bp; Positive control has obvious band at the arbitrary place of 199bp, 499bp or 330bp; Experiment is set up.Test sample occurs that 199bp amplified band is that Porcine epidemic diarrhea virus is positive, occur that 499bp amplified band is that transmissible gastro-enteritis virus is positive, occur that 330bp amplified band is that pig A rotavirus is positive, occurs that 2 or 3 amplified bands are then corresponding virus mixed infection, otherwise be feminine gender.
Test 2 specificity researchs
The Porcine epidemic diarrhea virus preserved this laboratory with test kit of the present invention, transmissible gastro-enteritis virus, pig A rotavirus, Pestivirus suis, porcine reproductive and respiratory syndrome virus, PRV (Pseudorabies virus) and pig circular ring virus carry out RT-PCR detection.
The specificity experiments of this test kit is shown: this test kit has very high specificity, Porcine epidemic diarrhea virus, between transmissible gastro-enteritis virus and pig A rotavirus, there is not cross reaction, and with other viruses as Pestivirus suis, porcine reproductive and respiratory syndrome virus, PRV (Pseudorabies virus), pig circular ring virus also equal no cross reaction.As Fig. 1 and Fig. 2.
Test 3 repetitive research
The Porcine epidemic diarrhea virus vaccine strain this laboratory preserved with test kit of the present invention and LPS strain, transmissible gastro-enteritis virus vaccine strain and LPS strain, pig A rotavirus vaccine strain and LPS strain.As Fig. 3.
The repeated experiment of this test kit is shown: this test kit has good repeatability, except detecting except vaccine strain, also can detect the Porcine epidemic diarrhea virus of different sources, transmissible gastro-enteritis virus and pig A rotavirus.
Test 4 Code's Sensitivities
Gradient dilution detection is carried out respectively with test kit extraction Porcine epidemic diarrhea virus of the present invention, transmissible gastro-enteritis virus, pig A rotavirus.As Fig. 4, Fig. 5 and Fig. 6.
The sensitivity experiments of this test kit is shown: this test kit has higher susceptibility, the extraction RNA concentration of Porcine epidemic diarrhea virus is, minimal detectable concentration is, the minimal detectable concentration of transmissible gastro-enteritis virus is be with the minimal detectable concentration of pig A rotavirus.
Annotate in figure:
Fig. 1 is the One step RT-PCR detected result that the present invention carries out PEDV, TGEV and GAR;
M:DL2000; 1:PEDV cell culture; 2:TGEV cell culture; 3:GAR cell culture; 4:PEDV and TGEV mixed cell culture thing; 5:PEDV and GAR mixed cell culture thing; 6:TGEV and GAR mixed cell culture thing; 7:PEDV, TGEV and GAR mixed cell culture thing.
Fig. 2 is the specific detection result that the present invention carries out PEDV, TGEV, GAR;
M:DL2000; 1:TGEV; 2:PEDV; 3:GAR; 4:CSFV; 5:PRRSV; 6:PRV; 7:PCV; 8: negative control.
Fig. 3 is the repeatable detected result that the present invention carries out PEDV, TGEV, GAR;
M:DL2000; 1: negative control; 2:PEDV vaccine strain; 3:PEDV LPS strain; 4:GAR vaccine strain; 5:GAR LPS strain; 6:TGEV vaccine strain; 7:TGEV LPS strain.
Fig. 4 is the susceptibility detected result that the present invention carries out PEDV;
M:DL2000; 1:PEDV; 2:PEDV 10
-1dilution; 3:PEDV 10
-2dilution; 4:PEDV 10
-3dilution; 5:PEDV 10
-4dilution; 6:PEDV 10
-5dilution; 7:PEDV 10
-6dilution; 8:PEDV 10
-7dilution; 9:PEDV 10
-8dilution; 10: negative control.
Fig. 5 is the susceptibility detected result that the present invention carries out TGEV;
M:DL2000; 1:TGEV; 2:TGEV 10
-1dilution; 3:TGEV 10
-2dilution; 4:TGEV 10
-3dilution; 5:TGEV 10
-4dilution; 6:TGEV 10
-5dilution; 7:TGEV 10
-6dilution; 8:TGEV 10
-7dilution; 9:TGEV 10
-8dilution; 10: negative control.
Fig. 6 is the susceptibility detected result that the present invention carries out GAR;
M:DL2000; 1:GAR; 2:GAR 10
-1dilution; 3:GAR 10
-2dilution; 4:GAR 10
-3dilution; 5:GAR 10
-4dilution; 6:GAR 10
-5dilution; 7:GAR 10
-6dilution; 8:GAR 10
-7dilution; 9:GAR 10
-8dilution; 10: negative control.
Clinical data
1, as the test kit detecting Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig A rotavirus, select the routine clinical observation of patient 200 of making a definite diagnosis, patient is divided into test set and control group two groups at random, test set 100 example, 35 ~ 55 years old age, 40.8 years old mean age.Control group 100 example, 35 ~ 55 years old age, 40.1 years old mean age.Be mixed into respectively in test set and control group and do not suffer from patient 100 people.
The course of disease, the degrees of symptoms of test set and control group two groups of cases are basically identical, without significant difference, have comparability.
2, detection method
Test set adopts test kit of the present invention to detect;
Control group adopts ordinary method to detect.
3, select test kit of the present invention to detect, only need within 4.5 hours, can detect result, accuracy rate is up to 100%.
Control group detects needs more than 8 hours, and accuracy rate is 94%.
Above result display, test set required time shortens greatly, and accuracy rate is higher.
Model case is illustrated: Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig A rotavirus,
, Wang, man, 48 years old, suffer from Porcine epidemic diarrhea virus, adopt test kit of the present invention to detect, 10 AM detection, namely there is accurate detected result at 2 in afternoon.
, Zhang, man, 44 years old.Affected pig Transmissible gastroenteritis virus, adopts test kit of the present invention to detect, detection at ten one in the morning, and namely accurate detected result appears in 3: 30 afternoon.
, Wang, man, 37 years old.Affected pig A rotavirus, adopts test kit of the present invention to detect, and a bit detect in the afternoon, and namely accurate detected result appears in 5 PM 20 points.
SEQUENCE LISTING
<110> Luoyang Lai Pusheng Information technology company limited
<120> triple RT-PCR detection kit and detection method thereof
<130>
<160> 6
<170> PatentIn version 3.3
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<212> DNA
<213> synthetic
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tttattctgt tacgccatgt 20
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<212> DNA
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ccagatttac aaacacctat gtta 24
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<212> DNA
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tttgacttgc aattggggta g 21
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<212> DNA
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aaagctaggg actggccata a 21
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<212> DNA
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ccgcggtatt gtatatagca cagt 24
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<212> DNA
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tttgtgttcg ccagccatta gt 22
Claims (1)
1. a triple RT-PCR detection kit, it is characterized in that: this test kit is formed by the A box extracted for viral RNA with for the B box that RT-PCR detects, be provided with damping fluid RG 9 mL, damping fluid RF 3 mL, damping fluid RD 8 mL, damping fluid RW 12 mL, RNA elutriant 3 mL, 50 times of TAE electrophoretic buffer 8 mL, staining fluid 10 μ L, sample-loading buffer 30 μ L, Rnase-free adsorption column, collection tube 10, centrifuge tube 10 and 0.2 mL PCR pipe 10 in A box, in B box, be provided with amplification reaction solution 220 μ L, enzyme system 10 μ L; Described amplification reaction solution comprises the PCR primer sets for detecting Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig A rotavirus, described primer sets is made up of three groups of primer sequences, and the primer sequence detected for Porcine epidemic diarrhea virus is respectively SEQ ID NO:1 and SEQ ID NO:2; The primer sequence detected for transmissible gastro-enteritis virus is respectively SEQ ID NO:3 and SEQ ID NO:4; The primer sequence detected for pig A rotavirus is respectively SEQ ID NO:5 and SEQ ID NO:6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510146276.5A CN104789698A (en) | 2015-03-31 | 2015-03-31 | Triple RT-PCR detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510146276.5A CN104789698A (en) | 2015-03-31 | 2015-03-31 | Triple RT-PCR detection kit |
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| CN104789698A true CN104789698A (en) | 2015-07-22 |
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| CN201510146276.5A Pending CN104789698A (en) | 2015-03-31 | 2015-03-31 | Triple RT-PCR detection kit |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409558A (en) * | 2013-07-10 | 2013-11-27 | 东北农业大学 | Multiplex PCR primer group for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus simultaneously |
| CN104293977A (en) * | 2014-09-29 | 2015-01-21 | 四川农业大学 | Gene chip and kit for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus |
-
2015
- 2015-03-31 CN CN201510146276.5A patent/CN104789698A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409558A (en) * | 2013-07-10 | 2013-11-27 | 东北农业大学 | Multiplex PCR primer group for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus simultaneously |
| CN104293977A (en) * | 2014-09-29 | 2015-01-21 | 四川农业大学 | Gene chip and kit for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus |
Non-Patent Citations (2)
| Title |
|---|
| 张坤: "猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪A群轮状病毒多重RT-PCR检测方法的建立及临床应用", 《畜牧兽医学报》 * |
| 郑新添: "猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒三重PCR检测方法的建立", 《龙岩学院学报》 * |
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