CN103952487B - Catch-dual real-time fluorescence PCR detection method of the magnetic of campylobacter jejuni and salmonella - Google Patents
Catch-dual real-time fluorescence PCR detection method of the magnetic of campylobacter jejuni and salmonella Download PDFInfo
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Abstract
The method that catch-dual real-time fluorescence PCR of the magnetic that the invention discloses a kind of campylobacter jejuni and salmonella detects, the method utilizes antiserum and magnetic micro-beads to prepare campylobacter jejuni and salmonella immunomagnetic beads, utilize object bacterium in immunomagnetic beads Direct Acquisition sample, cultivate without increasing bacterium; The present invention utilizes dual real-time fluorescence PCR method, design respectively Auele Specific Primer and the detector probe of the conservative gene of campylobacter jejuni and salmonella, increase whether be subject to the pollution of campylobacter jejuni and salmonella in judgement sample by a real-time fluorescence PCR; The inventive method is simple and easy to do, can within a working day, complete, and specificity is good, and susceptibility is high, is a kind of rapid technology method that is applicable to the fields such as inspection and quarantine, prevention from suffering from the diseases and animal production safety inspection.
Description
Technical field
The invention belongs to inspection and quarantine field, relate to dual real-time fluorescence PCR detection method, be specifically related to a kind of application and exempt fromEpidemic disease magnetic bead technology is carried out the method for catch-dual real-time fluorescence PCR of the magnetic detection of campylobacter jejuni and salmonella.
Background technology
Campylobacter jejuni and salmonella are the common pathogens that causes food posioning, often pollute meat, breast andThe animal foods such as fowl, the edible food polluting can cause the acute gastroenteritis symptoms such as diarrhoea, vomiting, serious causing is deadDie. Campylobacter jejuni is the pandemic Zoonosis cause of disease of the world wide bacterium of being familiar with in recent ten years, world healthOrganize and this disease is classified as to one of modal food source sexually transmitted disease. The infection of campylobacter jejuni also with people's guillain-BarreSyndromes etc. have disease relevant (McCarthyN, GieseckeJ, WeinbergED, the et of autoimmunity characteristical.IncidenceofGuillainBarreSyndromefollowinginfectionwithCampylobacterjejuni[J] .AmJEpidemiol, 2001,153 (6): 610-614). According to China and the worldThe statistics of various countries shows, the food poisoning that salmonella causes occupies first place in food posioning, now becomesThe major issue of food public health aspect.
The initial stage symptom of campylobacter jejuni and salmonella infection is very similar, and this has caused tired to distinguishing two kinds of diseasesDifficult. To the detection of campylobacter jejuni and salmonella, traditional cultural method comprises increasing bacterium, Physiology and biochemistry qualification, serologyThe operations such as qualification, but required Check-Out Time, quantity long and that pathogen exists in food samples was relatively less, used normalThe method of the selective dull and stereotyped upper suspicious bacterium colony of picking of rule also may cause the undetected of positive strain. It is micro-that campylobacter jejuni belongs toAerobic bacteria, to culture medium and cultivate gaseous environment require all stricter, and the separation of bacterium cultivate waste time and energy, work as infectionOr the amount of polluting is when less, isolates campylobacter jejuni just more difficult from sample. It is reported, the causing of campylobacter jejuniSick dosage is very little, and 400～500 thalline just can cause enteric infection (MandellGL, BennettJE, Dolin, etal.Principlesandpracticeofinfectiousdiseases4thedition[M].NewYorkChurchillLivingstone, 1995,2053 – 2060.), therefore set up the detection method of high sensitivity and high specificityThe important measures that improve campylobacter jejuni recall rate. In addition, this bacterium enters by Host animals person or trouble patient's ight soilIn environment, can in water environment, enter one " live non-cultivation " (viablebutnon-culturerable,VBNC) state (MckayAM.Viablebutnon-culturableformsofpotentiallypathogenicbacteriainwater[J].LettApplMicrobiol,1992,14:129-135.MedemaGJ,SchetsFM,VandeGiessenAW,etal.Lackofcolonizationof1dayoldchicksbyviable,non-culturableCampylobacterjejuni[J].JApplBacteriol, 1992,75:512-516.), the bacterium in VBNC state can recover under given conditions andHave pathogenic. This difficult problem that tradition separation cultivation detection method cannot overcome especially. Therefore in the inspection of campylobacter jejuniIn quarantine, be badly in need of a kind of sensitive method for quick.
The false positive results appearance that some simple methods are as higher in PCR, ELISA often have, therefore its result all can notAs final detection foundation. Along with Protocols in Molecular Biology fast development, it is micro-that real-time fluorescence PCR technology is widely used in cause of diseaseBiological detection. In real-time fluorescence PCR reaction, introduce a kind of fluorescence chemical material, PCR product is carried out to mark tracking. WithThe carrying out that PCR reaction, PCR product is constantly accumulated, and fluorescence signal intensity also equal proportion increases. Be every through oneCirculation, fluorescence monitoring system can be collected a fluorescence intensity signals, thereby has realized accumulation and the formation of PCR product of fluorescence signalComplete Synchronization, real time and on line monitoring reaction overall process. Thereby monitor the variation of product amount by fluorescence intensity change, obtain oneAmplified fluorescence curve map, then can also analyze product in conjunction with corresponding software, calculates the initial of testing sample templateConcentration. Compare with conventional PCR, real-time fluorescence PCR have specificity stronger, can effectively solve that PCR pollutes, automaticity is high andRealize the advantage such as real-time monitoring of PCR reaction, be used widely at present.
1979, JohnUgelstad etc. successfully prepared a kind of uniformity and the suitable polystyrene microsphere of granularity,After its magnetization being connected with antibody, become the splendid immunomagnetic beads-Dynabeads of a kind of isolated cell effect. From then on, exempt fromEpidemic disease magnetic bead is used widely, and has caused the revolution on bioseparation technology.
Immunomagnetic bead technique (immunomagneticbeadstechniques, IMB) is a kind of utilization through modifyingMagnetic microsphere carry out as carrier that object is caught and the technology of enrichment. The key point of this technology is specific immunomagnetic beadsPreparation. Magnetic bead, after certain processing, can, in conjunction with the specific antibody of certain microorganism, form immunomagnetic beads, by this bandHave the immunomagnetic beads of specific antibody to be added in testing sample, corresponding antigenic substance will with immunomagnetic beads on antibody occurSpecific binding, forms Ag-Ab-magnetic bead immune complex. This compound occurs under stronger externally-applied magnetic field effectMechanics moves, and makes compound and other separating substances, thereby reaches the object of quick separation antigenic substance. Then, remove magnetic field,Collect magnetic bead, antigenic substance is disintegrated down it is identified from magnetic bead, or directly carry out the inspections such as PCR or fluorescent stainingTest. This technology is widely used in separation and purification and the microorganism detection field etc. of biogenetic products in recent years, especially micro-in cause of diseaseThe application of bio-separation context of detection is increasingly extensive, has developed for the commercialization immunomagnetic beads of various pathogenic bacteria and has detected examinationAgent box, and be progressively applied in the coherent detection work in laboratory.
Summary of the invention
Binding immunoassay magnetic bead technology of the present invention and dual real-time fluorescence PCR technology, set up a kind of jejunum campylobacter bar firstCatch-dual real-time fluorescence PCR detection method of the magnetic of bacterium and salmonella, for realizing the same of campylobacter jejuni and salmonellaIn time, is detected new method is provided.
The object of the present invention is to provide the magnetic of a kind of campylobacter jejuni and salmonella to catch-dual real-time fluorescencePCR detection method.
The technical solution used in the present invention is:
Catch-dual real-time fluorescence PCR detection method of the magnetic of campylobacter jejuni and salmonella, comprises the following steps:
(1) preparation of immunomagnetic beads:
1) produce respectively antibodies toward salmonella and campylobacter jejuni antibody;
2) magnetic bead is carried out to oscillation incubation with above-mentioned antibodies toward salmonella and campylobacter jejuni antibody respectively, make antibody withThe abundant combination of magnetic bead;
3), by the magnetic bead washes clean of hatching, 4 DEG C of preservations, can obtain respectively exempting from of salmonella and campylobacter jejuniEpidemic disease magnetic bead;
(2) magnetic of object bacterium is caught
1) by centrifugal sample to be checked, abandon supernatant, precipitation is resuspended in the PBS buffer solution containing BSA;
2) get above-mentioned suspension to be checked and add the examination containing campylobacter jejuni immunomagnetic beads and salmonella immunomagnetic beads suspensionGuan Zhong, room temperature oscillation incubation makes object bacterium be combined completely with immunomagnetic beads, discards liquid; Add again the PBS buffer solution containing BSAClean up in connection with the immunomagnetic beads that has object bacterium;
3) add deionized water to mix the immunomagnetic beads of above-mentioned washes clean, for extracting DNA of bacteria;
(3) dual real-time fluorescence PCR:
1) preparation of DNA of bacteria template: by above-mentioned magnetic bead of catching object bacterium in 95～100 DEG C of cracking, ice bath 10～15min, then, through centrifuging and taking supernatant, is template DNA;
2) template DNA of above-mentioned acquisition is carried out to dual real-time fluorescence PCR reaction, reaction system is:
2.5 × RealMasterMix (containing internal reference dyestuff ROX), 8 μ L,
Primers F-1(10 μ M) 1 μ L
Primer R-1(10 μ M) 1 μ L
Primers F-2(10 μ M) 1 μ L
Primer R-2(10 μ M) 1 μ L
Detector probe-1(10 μ M) 0.3 μ L
Detector probe-2(10 μ M) 0.7 μ L
20×probeEnhancersolution1μL
Template DNA 2 μ L
DdH2O mends to 20 μ L;
Wherein, primers F-1, primer R-1, detector probe-1 be the primer that detects of campylobacter jejuni real-time fluorescence PCR andProbe, its sequence is as follows:
Primers F-1:5'TGCTGAAGAGGGTTTGGGT-3'
Primer R-1:5'ACCCCTTCCAATAACTTCAATACT-3'
Detector probe-1:5'FAMTCCGAAGAAGCCATCATCGCACCTAMRA-3'
The 5' end of campylobacter jejuni detector probe sequence is marked with fluorescence report group FAM, and 3' end is marked with fluorescence and quenchesGroup TAMRA goes out;
Primer and probe that primers F-2, primer R-2, detector probe-2 are detected for salmonella real-time fluorescence PCR, its orderBe listed as follows:
Primers F-2:5'-ATTTGTATTGGTTGTTACGGCTATT-3'
Primer R-2:5'-TGCTCGCCTTTGCTGGTT-3'
Detector probe-2:5'JOE-TTCAATGGGAACTCTGCCGGGATT-TAMRA3'
The 5' end of salmonella detector probe sequence is marked with fluorescence report group JOE, and 3' end is marked with fluorescent quenching baseThe TAMRA of group;
3) interpretation of result and judgement: after reaction finishes, adjust threshold value, observe negative control product, its FAM, JOE fluorescence signalValue does not have significant change, amplification Ct value not or more than 40, can judge that this experiment is reliable; Detect in sample, if FAM is glimmeringLight is collected the S-type amplification curve of signal and Ct value < 35, and this sample is the campylobacter jejuni positive, if should in 35 < Ct value < 40Sample is reformed; If Ct value > 40, this sample is campylobacter jejuni feminine gender; Detect in sample, if JOE phosphor collection signal is SType amplification curve and Ct value<35, be the salmonella positive, if 35<Ct value<40, this sample is reformed; If Ct value>40, beSalmonella feminine gender.
Further, above-mentioned antibodies toward salmonella is Salmonella A-F group diagnostic serum.
Further, the reaction condition of above-mentioned dual real-time fluorescence PCR is: 95 DEG C of denaturation 2min, and 95 DEG C of sex change 20s,60 DEG C of 1min, 45 circulations.
The invention has the beneficial effects as follows:
1) binding immunoassay magnetic bead technology of the present invention and dual real-time fluorescence PCR technology, set up a kind of jejunum campylobacter firstCatch-dual real-time fluorescence PCR detection method of the magnetic of bacillus and salmonella, be a kind of easy, quick, specificity is high, sensitiveSpend the strong detection method that can simultaneously detect campylobacter jejuni and salmonella, by a real-time fluorescence PCR amplificationIn judgement sample, whether be subject to the pollution of campylobacter jejuni and salmonella.
2) the Bacteria Detection sensitivity of the inventive method to non-culmrable sate (VBNC), for improving food microorganisms inspectionAccuracy, improve bacterium picking out rate, the sanitary condition of correctly evaluating food has important meaning.
3) the present invention's application antiserum and magnetic micro-beads are prepared campylobacter jejuni and salmonella immunomagnetic beads, profit voluntarilyWith the object bacterium in immunomagnetic beads Direct Acquisition testing sample, testing process does not need to increase bacterium cultivates, simple and easy to do, can be at oneIn working day, complete; The present invention utilizes dual real-time fluorescence PCR method, according to the conservative base of campylobacter jejuni and salmonellaThe conservative region of cause designs respectively synthetic primer and detector probe, gets final product in judgement sample by a real-time fluorescence PCR amplificationWhether be subject to the pollution of campylobacter jejuni and salmonella, and specificity is good, susceptibility is high, be one be applicable to inspection and quarantine,The rapid technology method in the fields such as prevention from suffering from the diseases and animal production safety inspection.
Brief description of the drawings
Fig. 1 is catch-dual real-time fluorescence PCR of magnetic specific amplification curve; Black curve is that 2 strain campylobacter jejunis expandIncrease curve; Purple curve is 2 strain salmonella amplification curves;
Fig. 2 is that catch-dual real-time fluorescence PCR of magnetic is to campylobacter jejuni sensitivity amplification, song from left to rightThe concentration of line is followed successively by: 1.0 × 106CFU/mL，1.0×105CFU/mL，1.0×104CFU/mL，1.0×103CFU/mL，1.0×102CFU/mL，1.0×101CFU/mL；
Fig. 3 be catch-dual real-time fluorescence PCR of magnetic to salmonella sensitivity amplification, curve is from left to right denseDegree is followed successively by: 2.1 × 106CFU/mL，2.1×105CFU/mL，2.1×104CFU/mL，2.1×103CFU/mL，2.1×102CFU/mL；
Fig. 4 is the testing result of catch-dual real-time fluorescence PCR of magnetic to analog sample, and wherein, purple curve is two songsLine, because the amount of DNA profiling approaches, so curve almost overlaps, sample 1 is for adding the sample of campylobacter jejuni, and sample 2 is for addingAdd the sample of salmonella, sample 3 is for adding the sample of two kinds of bacteriums, the negative contrast of sample 4;
Fig. 5 is catch-dual real-time fluorescence PCR of magnetic sample detection amplification, and black curve is that campylobacter jejuni expandsIncrease curve; Purple curve is salmonella amplification curve.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to this.
Catch-the dual real-time fluorescence PCR detection method of magnetic of embodiment 1 campylobacter jejuni and salmonella
(1) preparation of salmonella immunomagnetic beads and campylobacter jejuni immunomagnetic beads
1) prepare respectively antibodies toward salmonella and campylobacter jejuni antibody;
Antibodies toward salmonella is selected Salmonella A-F group diagnostic serum, and (Ningbo Tian Run Bioceuticals Inc. producesProduct); Shown in being prepared as follows of campylobacter jejuni antibody:
A. the preparation of campylobacter jejuni antigen
Under aseptic condition, by campylobacter jejuni strains A TCC33291, NCTC11168, the ZC8 of-80 DEG C of freezing preservations,Respectively picking a little be inoculated on Colombia's blood agar under 42 DEG C, micro-aerobic condition and cultivate 36～48h. Continuous Cultivation goes down to posterity threeAfter inferior, observe the rear thalline situation of campylobacter jejuni recovery.
Add appropriate PBS(pH7.4) bacterium colony is washed, with the centrifugal 5min of 8000rpm, abandon supernatant, use PBS with phaseWith method wash thalline 3 times, then will precipitation with containing the PBS(pH7.4 of concentration of formaldehyde 2mL/L) be adjusted to bacterial concentration109CFU/ml, 37 DEG C of deactivation 16h. Through the inactivated bacterial liquid being up to the standards, get three kinds of deactivations of equivalent after bacterium liquid mix, asThe immunogene that inoculation new zealand rabbit is used, simultaneously also former as the reaction of test tube agglutination, 4 DEG C of Refrigerator stores are for subsequent use.
B. the emulsification of campylobacter jejuni antigen
Get appropriate inactivated bacterial liquid and mix in the ratio of 1:1 with Freund's complete adjuvant or incomplete Freund's adjuvant, with 22000Rpm high-speed stirred 30min, is oil emulsion inactivated vaccine. Get the emulsion inactivated vaccine that a dripping gets ready and drip in cold water surface,Bubble through the water column if keeping complete does not fall apart, become to drip a shape, emulsification success is described, otherwise it is successful to emulsification to continue high-speed stirred. 4 DEG C of guarantorsDeposit for subsequent use.
C. the preparation of campylobacter jejuni polyclonal antibody
The adult new zealand rabbit that body weight is about to 2kg by the immune programme for children shown in table carries out immunity, and immunity is for the first time usedFreund's complete adjuvant, all the other all use incomplete Freund's adjuvant, as shown in table 1 below. Immunity vestibule edge venous blood sampling 2mL separation of serumAs negative control. 2 weeks, each immune interval, 7d ~ 10d after immunity for the third time, ear vein blood sampling, separation of serum. Solidifying with test tubeCollection test detects tiring of serum, reaches 1:320 or when above when tiring, heart blood sampling, and separation of serum, if it is too low to tireCan exempt from one week post dose three and double booster immunization once.
The immune programme for children of table 1 rabbit
D. the purifying of campylobacter jejuni polyclonal antibody
1. by the serum being separated at 4 DEG C with the centrifugal 15min of 12000rpm, to remove impurity.
2. get the rabbit anteserum after centrifugal, add the 0.06mol/mL acetate buffer solution (PH4.0) of 3 times of volumes, the rear 5mol/ that usesThe Na0H of mL adjusts pH to 4.5. Under room temperature, stir 30min, slowly add during this time caprylic acid, its addition is by the front serum volume of dilutionCalculate 40 μ L/mL.
3. more than 4 DEG C of standing 2h, treat that it fully precipitates. Then the centrifugal 30min of 12000rpm, abandons precipitation.
4. the PBS (pH7.4) that adds l/10 volume in supernatant, is placed in ice bath environment.
5. in ice bath, slowly add isopyknic saturated ammonium sulfate, make it become 50% saturation degree. Keep magnetic agitation30min then (preferably spends the night) more than 4 DEG C of standing 2h, and it is fully precipitated.
6. the centrifugal 30min of 12000rpm under 4 DEG C of conditions, abandons supernatant.
7. be precipitated and dissolved in appropriate pH7.4,0.01mol/mLPBS buffer solution, moved in bag filter two endsTighten, bag filter be immersed in large beaker, and with the 0.01mol/mLPBS of 50-100 times of volume at 4 DEG C of dialysis 48h, every 6hChange dislysate one time, in dialysis procedure, use 2%BaCI2Solution detects in dislysate and has or not ammonium sulfate, after definite not liquid containing ammonium sulfate, and 4DEG C centrifugal 10min of 12000rpm, supernatant is how anti-that purifying obtains, and packing is frozen in-70 DEG C.
2) magnetic bead is combined with above-mentioned antibodies toward salmonella, campylobacter jejuni antibody respectively;
1. get respectively the 0.1mL magnetic bead liquid (DynabeadsM-280Sheepanti-that Dynal company producesRabbitIgG) in containing 1.0mLPBS(containing 0.1%BSA) in the 1.5mL centrifuge tube of buffer solution, mix; Be placed in magnetic frameUpper, leave standstill 2min, discard suspension; Adding 1.0mLPBS(to contain 0.1%BSA) buffer solution makes magnetic bead wherein resuspended;
2. add respectively 0.1mL campylobacter jejuni polyclonal antibody, Salmonella A-F group diagnostic serum (sky, NingboProfit Bioceuticals Inc. product), 37 DEG C of light and slow oscillation incubation 1h; Make the abundant combination of antibody and magnetic bead.
3) magnetic bead that is combined with antibody after hatching is placed on magnetic frame, leaves standstill 2min, remove liquid, with PBS(containing 0.1%BSA), after buffer solution for cleaning magnetic bead 3 times, magnetic bead is resuspended in to 1.0mLPBS(containing 0.1%BSA) in buffer solution, can obtain respectivelyObtain campylobacter jejuni immunomagnetic beads suspension, salmonella immunomagnetic beads suspension, 4 DEG C save backup.
(2) magnetic of object bacterium is caught
1) get 1.0mL bacteria suspension or the centrifugal 5min of sample 8000rpm to be checked, abandon supernatant liquid, precipitation is resuspended in 1.0mLPBS(is containing 0.1%BSA) in buffer solution;
2) get above-mentioned bacteria suspension in 0.2mL campylobacter jejuni immunomagnetic beads and 0.2mL salmonella immunomagnetic beads are housedIn the test tube of suspension, the light and slow rotational oscillation 1h of room temperature, is placed in 2min on magnet stand by tubule, carefully discards the liquid in pipe;
3) adding 1.0mLPBS(to contain 0.1%BSA) buffer solution mixes, and is placed in 2min on magnet stand, shifts out liquid, repeated washingAfter 3 times, add 0.1mL deionized water to mix, for extracting DNA of bacteria.
(3) dual real-time fluorescence PCR:
1) preparation of DNA of bacteria template: by above-mentioned magnetic bead of catching object bacterium in 100 DEG C of cracking 15min, ice bath10min then gets supernatant after the centrifugal 5min of 8000rpm, under-20 DEG C of conditions, saves backup;
2) primer and probe
The conservative gene hipO of campylobacter jejuni and the conservative gene invA sequence of salmonella announced according to NCBI,Utilize PrimerPremier5.0 and DNAStar Software for Design primer and TaqMan probe, the 5' end of probe is marked with glimmeringLight reporter group, 3' end is marked with fluorescent quenching group, and primer and probe are by Shanghai Ying Weijie base Bioisystech Co., LtdSynthetic. The sequence of primer and probe is as follows:
The primer that campylobacter jejuni real-time fluorescence PCR detects and the sequence of probe are as follows:
Primers F-1:5'TGCTGAAGAGGGTTTGGGT-3'
Primer R-1:5'ACCCCTTCCAATAACTTCAATACT-3'
Detector probe-1:5'FAMTCCGAAGAAGCCATCATCGCACCTAMRA-3'
The 5' end of campylobacter jejuni detector probe sequence is marked with fluorescence report group FAM, and 3' end is marked with fluorescence and quenchesGroup TAMRA goes out;
The primer that salmonella real-time fluorescence PCR detects and the sequence of probe are as follows:
Primers F-2:5'-ATTTGTATTGGTTGTTACGGCTATT-3'
Primer R-2:5'-TGCTCGCCTTTGCTGGTT-3'
Detector probe-2:5'JOE-TTCAATGGGAACTCTGCCGGGATT-TAMRA3'
The 5' end of salmonella detector probe sequence is marked with fluorescence report group JOE, and 3' end is marked with fluorescent quenching baseThe TAMRA of group.
3) dual real-time fluorescence PCR
Reaction system and the condition of model and optimization dual real-time fluorescence PCR, first do single mass system to every kind of pathogenQualitative reaction, is optimized primer, concentration and probe concentration and the Tm value of double fluorescent PCR reaction on this basis, sets up best anti-Answer system and amplification condition.
Best amplification condition is defined as: 95 DEG C of denaturation 2min, and 95 DEG C of sex change 20s, 60 DEG C of 1min, carry out 45 circulations.
Optimum response system (20 μ L) is: 2.5 × RealMasterMix (containing internal reference dyestuff ROX, dNTPs), 8 μ L, upper,((10 μ mol/L) is respectively 1 μ L to downstream primer, and probe (10 μ mol/L) is respectively 0.3 μ L (campylobacter jejuni), 0.7 μ L (sandDoor Salmonella), 20 × probeEnhancersolution1 μ L, template DNA 2 μ L, add ultra-pure water and complement to 20 μ L.
Through above-mentioned reaction system and amplification condition are optimized after, set up optimum dual real-time fluorescence PCR and be: willThe template DNA of above-mentioned acquisition carries out dual real-time fluorescence PCR reaction, and reaction system (20 μ L) is: 2.5 × RealMasterMix(containing internal reference dyestuff ROX) 8 μ L, ((10 μ mol/L) is respectively 1 μ L to various upstream and downstream primer, and two kinds of probes (10 μ mol/L) respectivelyBe 0.3 μ L (campylobacter jejuni), 0.7 μ L (salmonella), 20 × probeEnhancersolution1 μ L, mouldPlate DNA2 μ L, adds ultra-pure water and complements to 20 μ L. Reaction condition (two-step method) is: 95 DEG C of denaturation 2min, 95 DEG C of sex change 20s, 60DEG C 1min, 45 circulations;
Interpretation of result and judgement: after reaction finishes, adjust threshold value, observe negative control product, its FAM, JOE fluorescence signal valueThere is no significant change, increase Ct value not or more than 40, can judge that this experiment is reliable. Detect in sample, if FAM fluorescenceCollect the S-type amplification curve of signal and Ct value < 35, this sample is the campylobacter jejuni positive, if 35 < Ct value < 40, this sampleProduct are reformed; If Ct value > 40, this sample is campylobacter jejuni feminine gender. Detect in sample, if JOE phosphor collection signal is S-typeAmplification curve and Ct value<35, be the salmonella positive, if 35<Ct value<40, this sample is reformed; If Ct value>40, be huskyDoor Salmonella feminine gender.
The detection method of below the present invention being set up is made further effect detection.
One, specific test
The magnetic that listed 11 strains (9 kinds) Bacteria liquid in following table 1 is carried out respectively to campylobacter jejuni and salmonella catches-Dual real-time fluorescence PCR detects (detection method as described in Example 1), and its amplification is shown in Fig. 1 and table 1, and result shows 2 strain skiesIntestines campylobacter, 2 strain salmonellas all produce obvious amplification curve, show positive findings; And other 7 kinds of bacterial strains and deionizationsWater blank all can not produce amplification curve, shows negative findings. Illustrate that the method has good specificity. Through 3 timesRepeat test, result of the test is all consistent, shows good stability.
Table 1 test bacterial strain list
Note: "+" represents that testing result is positive; "-" represents that testing result is negative
Two, sensitivity test
Bacterial concentration is respectively to 1.0 × 106CFU/mL、2.1×106The campylobacter jejuni of CFU/mL and salmonellaBacterium liquid carries out respectively 10 times of gradient dilutions, gets respectively the each dilution liquid of 1.0mL and carries out campylobacter jejuni and salmonellaCatch-dual real-time fluorescence PCR of magnetic detect.
As shown in Figure 2, in figure, curve concentration is from left to right followed successively by campylobacter jejuni sensitivity test result: 1.0×106CFU/mL，1.0×105CFU/mL，1.0×104CFU/mL，1.0×103CFU/mL，1.0×102CFU/mL，1.0×101CFU/mL, therefrom can find out that detection method of the present invention can reach 10CFU/mL to the detectability of campylobacter jejuni.
As shown in Figure 3, in figure, curve concentration is from left to right followed successively by salmonella sensitivity test result: 2.1 ×106CFU/mL，2.1×105CFU/mL，2.1×104CFU/mL，2.1×103CFU/mL，2.1×102CFU/mL. Therefrom canFind out that detection method of the present invention can reach 210CFU/mL to the detectability of salmonella.
The above results shows that detection method of the present invention has highly the diagnosis of campylobacter jejuni and salmonellaSensitivity.
Three, the detection of analog sample
Gather frozen chicken meat sample, before artificial challenge, according to national standard method, sample is detected, and confirm itNot containing salmonella and campylobacter jejuni. Aseptic process sample is to rotten shape, accurately takes respectively 25g sample with homogenizing bag, pointDo not add 1mL finite concentration (106The CFU/mL order of magnitude) salmonella (sample 2), campylobacter jejuni (sample 1) and two kindsThe plastc ring of bacterium (sample 3), and establish the sample that does not add any bacteria suspension and make negative control (sample 4), then add225mL buffered peptone water (BPW) is in aseptic homogenizing bag, and vibration 30min, mixes it. Get a part of sample supernatantLiquid is in the centrifugal 2min of 3000rpm, and supernatant in the centrifugal 5min of 8000rpm, is abandoned liquid again, and precipitation is resuspended in 1.0mLPBS(is containing 0.1%BSA) buffer solution.
Above-mentioned sample is adopted respectively to campylobacter jejuni of the present invention and catch-dual real-time fluorescence of salmonella magneticPCR method detects, and its testing result as shown in Figure 4; To above-mentioned sample, according to national standard, " food hygiene is micro-respectively simultaneouslyBiological test salmonella inspection " standard method (GB/T4789.4-2010), " microbiological test of food hygiene jejunumCampylobacter inspection " standard method (GB/T4789.37-2008) detects campylobacter jejuni and salmonella, its inspectionSurvey the result consistent (as shown in table 2) that result detects with the inventive method, result is all shown as, to having added the mould of salmonellaIntend sample (sample 2) and amplify salmonella specific amplification curve, to having added the analog sample (sample of campylobacter jejuni1) amplify campylobacter jejuni specific amplification curve, the analog sample (sample 3) that adds these two kinds of bacteriums is increased simultaneouslyGo out the specific amplification curve of these two kinds of bacteriums, illustrate that the testing result of detection method of the present invention and national standard detection method is completeEntirely conform to.
Table 2 analog sample testing result
Note: "+" represents that testing result is positive; "-" represents that testing result is negative, and sample 1 is for adding jejunum campylobacter barThe sample of bacterium, sample 2 is for adding the sample of salmonella, and sample 3 is for adding the sample of two kinds of bacteriums, the negative contrast of sample 4.
Four, actual sample detects
Gather 30 parts of new fresh chicken meat class samples, adopt respectively the magnetic of campylobacter jejuni and salmonella to catch-dual realityTime fluorescence PCR method, traditional bacterium separate and cultivate and biochemical identification detection method, national standard method detect sample,And the testing result of 3 kinds of methods is compared.
Fig. 5 is the testing result of catch-dual real-time fluorescence PCR of the magnetic of campylobacter jejuni and salmonella method, knotFruit demonstrates 2 parts of campylobacter jejuni positive (sample 1 and 2, as shown in table 3), 5 parts of salmonella positive (samples 2~ 4, as shown in table 3), all the other total negatives, in full accord with the result that national standard Law detects, and separate with traditional bacteriumThe testing result of identification method has a positive difference (as shown in table 3), and Traditional Method can not detect the Salmonella in sample 2Bacterium. May be because salmonella content in sample 2 is fewer, the insufficient sensitivity of traditional detection method is high, easily causes false negative.The above results explanation the inventive method has good correctness and sensitivity.
The positive findings that the different detection methods of table 3 detect
Note: "+" represents that testing result is positive; "-" represents that testing result is negative
The inventive method confirms to have good repeatability and stability through the repeatedly different experiments that repeats.
Binding immunoassay magnetic bead technology of the present invention and dual real-time fluorescence PCR technology, set up a kind of jejunum campylobacter bar firstCatch-dual real-time fluorescence PCR detection method of the magnetic of bacterium and salmonella.
The method is because of the Bacteria Detection sensitivity to non-culmrable sate (VBNC) state, for improving food microorganisms inspectionAccuracy, improve bacterium picking out rate, the sanitary condition of correctly evaluating food has important meaning.
The inventive method is simple and easy to do, can within a working day, complete, and specificity is good, and susceptibility is high, is a kind of being suitable forIn the method for quick in the fields such as inspection and quarantine, prevention from suffering from the diseases and animal production safety inspection.
Claims (3)
1. catch-dual real-time fluorescence PCR detection method of the magnetic of campylobacter jejuni and salmonella, is characterized in that: compriseFollowing steps:
One, the preparation of immunomagnetic beads:
1) produce respectively antibodies toward salmonella and campylobacter jejuni antibody;
2) magnetic bead is carried out to oscillation incubation with above-mentioned antibodies toward salmonella and campylobacter jejuni antibody respectively, make antibody and magnetic beadFully combination;
3), by the magnetic bead washes clean of hatching, 4 DEG C of preservations, can obtain respectively the immune magnetic of salmonella and campylobacter jejuniPearl;
Two, the magnetic of object bacterium is caught
1) by centrifugal sample to be checked, abandon supernatant, precipitation is resuspended in the PBS buffer solution containing BSA;
2) get above-mentioned suspension to be checked and add in the test tube containing campylobacter jejuni immunomagnetic beads and salmonella immunomagnetic beads suspension,Room temperature oscillation incubation makes object bacterium be combined completely with immunomagnetic beads, discards liquid; Add again containing the PBS buffer solution of BSA in connection withThere is the immunomagnetic beads of object bacterium to clean up;
3) add deionized water to mix the immunomagnetic beads of above-mentioned washes clean, for extracting DNA of bacteria;
Three, dual real-time fluorescence PCR:
1) preparation of DNA of bacteria template: by above-mentioned magnetic bead of catching object bacterium in 95～100 DEG C of cracking, ice bath 10～15min, then, through centrifuging and taking supernatant, is template DNA;
2) template DNA of above-mentioned acquisition is carried out to dual real-time fluorescence PCR reaction, reaction system is:
Containing 2.5 × RealMasterMix8 μ L of internal reference dyestuff ROX,
10 μ M primers F-11 μ L
10 μ M primer R-11 μ L
10 μ M primers F-21 μ L
10 μ M primer R-21 μ L
10 μ M detector probe-10.3 μ L
10 μ M detector probe-20.7 μ L
20×probeEnhancersolution1μL
Template DNA 2 μ L
ddH2O mends to 20 μ L;
Wherein, primer and probe that primers F-1, primer R-1, detector probe-1 are detected for campylobacter jejuni real-time fluorescence PCR,Its sequence is as follows:
Primers F-1:5'TGCTGAAGAGGGTTTGGGT-3'
Primer R-1:5'ACCCCTTCCAATAACTTCAATACT-3'
Detector probe-1:5'FAMTCCGAAGAAGCCATCATCGCACCTAMRA-3'
The 5' end of campylobacter jejuni detector probe sequence is marked with fluorescence report group FAM, and 3' end is marked with fluorescent quenching baseThe TAMRA of group;
Primers F-2, primer R-2, detector probe-2 are primer and the probe that salmonella real-time fluorescence PCR detects, its sequence asUnder:
Primers F-2:5'-ATTTGTATTGGTTGTTACGGCTATT-3'
Primer R-2:5'-TGCTCGCCTTTGCTGGTT-3'
Detector probe-2:5'JOE-TTCAATGGGAACTCTGCCGGGATT-TAMRA3'
The 5' end of salmonella detector probe sequence is marked with fluorescence report group JOE, and 3' end is marked with fluorescent quenching groupTAMRA；
3) interpretation of result and judgement: after reaction finishes, adjust threshold value, observe negative control product, its FAM, JOE fluorescence signal value do not haveThere are significant change, amplification Ct value not to exist or more than 40, can judge that this experiment is reliable; Detect in sample, if FAM fluorescence is receivedThe collection S-type amplification curve of signal and Ct value < 35, this sample is the campylobacter jejuni positive, if 35 < Ct value < 40, this sampleReform; If Ct value > 40, this sample is campylobacter jejuni feminine gender; Detect in sample, if the S-type expansion of JOE phosphor collection signalIncreasing curve and Ct value<35, is the salmonella positive, if 35<Ct value<40, this sample is reformed; If Ct value>40, be sramanaSalmonella feminine gender;
Said method is for diagnosis and the treatment of non-disease.
2. catch-dual real-time fluorescence PCR of the magnetic of campylobacter jejuni according to claim 1 and salmonella detection sideMethod, is characterized in that: described antibodies toward salmonella is Salmonella A-F group diagnostic serum.
3. catch-dual real-time fluorescence PCR of the magnetic of campylobacter jejuni according to claim 1 and salmonella detection sideMethod, is characterized in that: the reaction condition of described dual real-time fluorescence PCR is: 95 DEG C of denaturation 2min, 95 DEG C of sex change 20s, 60DEG C 1min, 45 circulations.
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