CN101381767A - Universal real time fluorescent PCR detection method of trichinella - Google Patents
Universal real time fluorescent PCR detection method of trichinella Download PDFInfo
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Abstract
The invention provides a trichina general real-time fluorescent PCR detection method. Conserved region sequences of LS-rRNA genes of mitochondrion of various trichina isolated species are determined to be amplified target sequences; and a pair of special primers and a TaqMAN MGB probe are designed, synthesized and applied to general detection of trichina. As shown by a quantitative standard curve manufactured by positive quality-control standard products with different concentration gradients, the logarithm value and the Ct value of the quantitative die plate number with the different gradients have good relativity, and the correlation coefficient is 0.9984; simultaneously, the detection method has high sensitivity, and 132 copy numbers and 10<-5> trichinas can be detected at the lowest; as shown by detection of other parasites, health hosts and water contrast, the method has good specificity; and as shown by the repeated tests, the detection system provided by the invention has good stability, and the detection result has small variation and is controlled within the statistic range.
Description
(1) technical field
The present invention relates to a kind of pcr amplification primer and probe of each Trichinella spiralis isolated species plastosome Ls-rRNA gene fragment, and carry out the universal test method of each Trichinella spiralis isolated species simultaneously.
(2) background technology
Trichonematosis (Trichinosis) is that a kind of important people beast suffers from parasitosis altogether, mainly infects because of eating or eat half a lifetime the pork or other animal meats that contain trichinella larvae Nang Bao raw.Because the diversity of its host specificity extreme difference and transmission of pathogen approach, nearly all Mammals all can infect.No matter be that carnivorous, omnivore or worm food, phytophagous animal can infect, even some birds also can infect, the people also is one of susceptible host of Trichinella spiralis.By the end of so far, reported that at least more than 150 kind of Mammals can infect Trichinella spiralis, common have people, pig, mouse, dog, bear, fox, wolf, an ermine etc.
This disease was found over more than 100 year, did not only obtain control in full force and effect, and occurrence scope enlarges on the contrary gradually.This disease threatens very big to human health, can cause people's death because of myocarditis, pneumonia etc., the present nearly 1,100 ten thousand human infection persons in the whole world, and the World Health Organization (OIE) and the international trichonematosis council (ICT) have classified it as the disease that occurs once more.Classify pigs trichina disease as two class animal epidemics in " The Ministry of Agriculture of the People's Republic of China, MOA's animal epidemic prevention method ".Regulation in China's " meat hygiene inspection procedure " must be carried out trichinous quarantine by head when pig slaughtering, positive pig must destroy.Except that Australia and some island, its almost extend over the entire globe various places that distribute have become a kind of global disease of natural focus at present.This disease causes the tremendous economic loss to livestock industry (especially pig industry), meat product industry and foreign export etc., and traditional quarantine method loss is high, has had a strong impact on the international image of China's food sanitation safe.
In recent years, all point out according to genetics, biology and biochemical research and revolve hair and belong to and to contain a plurality of isolated speciess.Think that at present the Trichinella spiralis genus has 8 isolated speciess (isolate) at least, be trichina(Trichinella spiralis) (T.spiralis, T1), local hair shape nematode (T.nativa, T2), Bu Shi hair shape nematode (T.britovi, T3), pseudo-trichina(Trichinella spiralis) (T.pseudospiralis, T4), Michaelis hair shape nematode (T.murrelli, T5), Nissl hair shape nematode (T.nelsoni, T7), Papua hair shape nematode (T.papuae, T10), Zimbabwe hair shape nematode (T.zimbabwensis, T11) and 3 still undetermined genotype of classification position (genotype), i.e. T6, T8 and T9.The regional distribution of every kind of Trichinella spiralis, host, all inequality to the pathogenic effects of human body and biological characteristics etc.
At present, China has two kinds of Trichinella spiraliss, i.e. T.spiralis and T.nativa at least.
People have carried out research extensively and profoundly to the excretory-secretory antigen of Trichinella spiralis in recent years, and some diagnosis detecting methods have been set up based on ELISA, though sensitive and special characteristics with technology for detection trichonematosis tools such as ELISA, but majority is to use Detection of antigen antibody, yet whether always whether not consistent with the appearance of muscle larvae the existence of antibody sometimes is.Be reported in the artificial challenge Trichinella spiralis after 16～20 weeks, have muscle larvae alive to exist in the skeletal muscle, but detect less than antibody this moment, false negative result occurs.Directly detect circulating antigen (CAg) among the trichonematosis patients serum with antibody, recall rate is very low, only has 47% patient can measure CAg clinically.
Along with the development of Protocols in Molecular Biology, Chinese scholars is also being done a large amount of research aspect the gene diagnosis of Trichinella spiralis.Klassen etc. (1986) are with restriction enzyme EcoRI digestion Trichinella spiralis genomic dna the time, it is that its copy number of tumor-necrosis factor glycoproteins of 1.7Kb is 2 that discovery has a length, 800, in genome, be direct arranged in series, account for 2% of whole genome DNA, this fragment has the strain specificity to pig type Trichinella spiralis.Dupouy (1991) etc. according to this sequences Design one couple of PCR primers, the result can amplify the specific DNA of 602bp from two kinds of samples, its sensitivity can reach 2ngDNA (being equivalent to 0.02 muscle larvae).Zarlenga etc. (1991) screen a specificity tumor-necrosis factor glycoproteins PUPB-3.7 (482bp) from the DNA library of the forest wildlife type Trichinella spiralis worm strain of structure, as probe the DNA of 20 strain forest wildlife type Trichinella spiraliss is carried out molecular hybridization, there are 19 strains to be positive, and can detect the genomic dna of 0.4ng.Dubey etc. (1994) also identify deriving from the intravital Trichinella spiralis of black bear with this probe, prove that PUPB-3.7 has the strain specificity to forest wildlife type Trichinella spiralis.But the gene diagnosis of system research rarely has report, and does not still have the universal detection method of each Trichinella spiralis isolated species at present.
The real-time fluorescence PCR technology is the high-new detection technique with great development potentiality, obtained in a lot of fields using widely, this technology has not only realized the leap of PCR method from qualitative to quantitative, and compare with conventional PCR, it has, and specificity is stronger, sense cycle short, effectively solve PCR pollutes characteristics such as asking the level of automation height, has been widely used in fields such as basic scientific research, clinical diagnosis, disease research and medicament research and development.
Because the trichonematosis substance has a plurality of isolated speciess, conventional inspection and quarantine means loss height, subjective dependency to the tester is higher, so press for set up a kind of special sensitivity, accurately and reliably, fast and convenient each Trichinella spiralis isolated species " single stage method " universal test method, be used for rapid detection and import and export meat product, thereby satisfy the needs of importing and exporting inspection and quarantine and eqpidemic disease monitoring.
(3) summary of the invention
The object of the present invention is to provide a kind ofly have fast, accurately, the universal real time fluorescent PCR detection method of trichinella of high-throughput, highly sensitive, high specificity characteristics.
The object of the present invention is achieved like this: choose the target sequence of each Trichinella spiralis isolated species plastosome LS-rRNA gene conserved regions sequence as pcr amplification, synthetic specific primer of design and TaqMAN MGB probe carry out qualitative detection to Trichinella spiralis.
The present invention also has some technical characterictics like this:
1, the synthetic following primer of described setting to TaqMAN MGB probe:
P1：5’-TGCAGTCTTAAAGAGAATCCAACCT-3’，
P2：5’-CAAGTTTCGTCTGTTCGACGATA-3’，
Probe：5’-Fam-TTGCGACGGTTTAAA-NFQ-MGB-3’；
2, described Trichinella spiralis comprises trichina(Trichinella spiralis) (Trichinella spiralis, T1), local hair shape nematode (Trichinellanativa, T2), pig Trichinella spiralis Heilungkiang isolated species (Trichinella swine isolate from Heilongjiang), dog Trichinella spiralis Heilungkiang isolated species (Trichinella dog isolate from Heilongjiang), pig Trichinella spiralis U.S. isolated species (Trichinellaswine isolate from USA) detect;
3, described pcr amplification reaction reaction system is:
Aseptic double-distilled water 12 μ l
10 times of reaction buffers (10 *), 2.5 μ l
MgCl
2(25mM) 2.4μl
Thymus nucleic acid (dNTP) is 1.5 μ l (2.5mM)
Primer 1 (10 μ M) 0.5 μ l
Primer 2 (10 μ M) 0.5 μ l
TaqMAN?MGB?Probes(10μM) 0.3μl
Taq archaeal dna polymerase (5U/ μ l) 0.3 μ l
Reaction conditions is: 95 ℃ of pre-sex change 2min; 95 ℃ of sex change 10s, 30s is extended in 60 ℃ of annealing, and 40cycles collects fluorescent signal in the time of 60 ℃.
Specificity experiment shows that the inventive method can specificly detect each Trichinella spiralis isolated species, and probe does not have with other parasites and host and intersects; Sensitivity experiment shows, the linearity range broadness that the inventive method detects, and low energy detects 10
2Copy, even to 10
-5The bar Trichinella spiralis also can detect; No matter be repeated experiment between interior repeated experiment of group or group, the variation coefficient of Ct value is controlled within the statistics useful range, proves having good stability of the inventive method detected result.
Thinking of the present invention is on the basis of the Trichinella spiralis nucleotide sequence that landed in extensive query analysis NCBI, in conjunction with the information biology means, determine with Trichinella spiralis plastosome Ls-rRNA gene conserved regions sequence as the amplified target sequence, a pair of Auele Specific Primer has been synthesized in design, to each Trichinella spiralis isolated species DNA (trichina(Trichinella spiralis), local hair shape nematode, pig Trichinella spiralis Heilungkiang isolated species, dog Trichinella spiralis Heilungkiang isolated species and pig Trichinella spiralis U.S. isolated species) increase, and the nucleotide sequence of the part that will increase carries out the BLAST comparative analysis with reported sequence, designed the Taqman MGB probe that can hybridize with all Trichinella spiralis isolated speciess with Primer Express3.0 software at conservative region.
Conventional PCR product is connected the pMD-18-T carrier, through transforming, extract plasmid, quantitative, prepare the positive quality control standard substance, the multiple proportions serial dilution with 5 makes 5.15 * 10
7, 1.03 * 10
7, 2.06 * 10
6, 4.12 * 10
5, 8.24 * 10
4, 1.65 * 10
4, 3.3 * 10
3, 6.6 * 10
2, 1.32 * 10
2The positive quality control standard substance of copies/ μ l are got the standard substance (4.12 * 10 of Ct value about 20
5Copies/ μ l) positive template when setting up as detection architecture.Adopt the experiment of single-factor concentration gradient progressively to change each concentration of component of PCR reaction system, optimize the real-time fluorescence PCR experiment condition, and regulate loop parameter, set up two warm circulation real-time fluorescence PCR detection methods.
The present invention is respectively with trichina(Trichinella spiralis), local hair shape nematode, pig Trichinella spiralis U.S. isolated species, pig Trichinella spiralis Heilungkiang isolated species, dog Trichinella spiralis Heilungkiang isolated species genomic dna and ascaris suum, trichuris suis, the pig hookworm, ascaris alata, haemonchus contortus, toxoplasma gondii, healthy pork, dog meat, beef, the genomic dna of mouse muscle experimentizes, to probe into the specificity that this method detects, result only each Trichinella spiralis isolated species can detect amplification curve, and each health hosts of other parasites does not all have the increase of fluorescent signal, proves that this method has good detection specificity and versatility.
Respectively get 5.15 * 10
7, 1.03 * 10
7, 2.06 * 10
6, 4.12 * 10
5, 8.24 * 10
4, 1.65 * 10
4, 3.3 * 10
3, 6.6 * 10
2, 1.32 * 10
2The positive quality control standard substance 2 μ l of copies/ μ l, with no template water to complying negative control, carry out amplified reaction on the fluorescent PCR instrument, the result shows that there is tangible linear relationship (R in the logarithm that the fluorescent signal in each reaction tubes reaches required cycle number of threshold value (Ct) and starting template copy number
2=0.9984), data point and the fitting of a curve of positive quality control standard substance on typical curve is good, amplification equation Y=-3.43X+19.70, and amplification efficiency is 95%, 40 minimum sample that detects 132 copies of circulation real-time fluorescence PCR; With real-time fluorescence PCR it is detected after genomic dna of extraction wall scroll Trichinella spiralis and the serial dilution, the result shows that present method is to 10
-1, 10
-2, 10
-3, 10
-4With 10
-5The bar Trichinella spiralis can both well detect.The no template contrast of NTC (no template control) does not have the amplified fluorescence curve, and negative result illustrates that present method has high susceptibility.
Real-time fluorescence PCR repeats 20 times and detects same positive quality control standard substance, and the variation coefficient that detects gained Ct value is 1.11%; Twice (30d at interval) detects the positive quality control standard substance with a collection of different concns gradient, detects gained Ct value and do not have significant difference through comparing test of hypothesis, and visible present method has good repeatability and stable.
Adopt each Trichinella spiralis isolated species universal real time fluorescent PCR detection method of setting up, analog sample and actual sample are detected.By detecting the muscle sample of various dose pig Trichinella spiralis mouse such as artificial challenge 5 larvas/only, 10 larvas/only, 20 larvas/only and 40 larvas/only, find that real-time fluorescence PCR can detect each infective dose in two weeks after infection, recall rate is 100%.Compare with the artificial digestion method with traditional compressing tablet microscopy, real-time fluorescence PCR detection method has fast, accurately, high-throughput, highly sensitive, characteristics that specificity is high.Compare with colloidal gold technique, real-time fluorescence PCR is wide in range not as the former on the scope of detection time.63 dog meats samples to censorship detect, and the result is all negative shown in compressing tablet microscopy, artificial digestion and the colloidal gold strip, and real-time fluorescence PCR then therefrom detects 8 weak positive samples, proves that present method is more sensitive.
The invention provides a kind of each Trichinella spiralis isolated species universal real time fluorescent PCR detection method.Determine with each Trichinella spiralis isolated species plastosome LS-rRNA gene conserved regions sequence that as the amplified target sequence design has been synthesized a pair of Auele Specific Primer and TaqMAN MGB probe and has been applied to general detection to Trichinella spiralis.The quantitative criterion curve of making according to the positive quality control standard substance of different concns gradient has dependency preferably between the logarithmic value of different gradient quantitative templates numbers and the Ct value as can be seen, and its relation conefficient is 0.9984; Simultaneously, this detection method has very high sensitivity, and low energy detects 132 copy numbers and 10
-5The bar Trichinella spiralis; Other parasites and health hosts and water contrast detected show that this method has excellent specificity; Repeated experiment shows that detection architecture provided by the invention has good stability, and its detected result variation is less, is controlled within the statistics scope.
(4) description of drawings
Fig. 1 is that (wherein curve 1～5: each Trichinella spiralis isolated species for real-time fluorescence PCR detection technique specificity experimental control figure; 6～16: negative sample and the contrast of no specimen water);
Fig. 2 for the kinetic curve of different copy number positive quality control standard substance fluorescent PCR amplifications (is: 5.15 * 10 from left to right successively
7, 1.03 * 10
7, 2.06 * 10
6, 4.12 * 10
5, 8.24 * 10
4, 1.65 * 10
43.3 * 10
3, 6.6 * 10
2, 1.32 * 10
2Copies/ μ l);
Fig. 3 is the real-time fluorescence PCR typical curve;
Fig. 4 is that wall scroll Trichinella spiralis genomic dna real-time fluorescence PCR detects contrast figure (curve 1～5:10 wherein
-1, 10
-2, 10
-3, 10
-4, 10
-5The bar Trichinella spiralis; CK: negative control).
(5) embodiment
The present invention is further illustrated below in conjunction with specific embodiment:
1.PCR the preparation of template: the extraction and the purifying that carry out DNA with reference to the sky for epoch TIANamp Genomic DNAKit specification sheets.Add all ingredients in proportion.
1.1 get an amount of detected sample, add 200 μ l damping fluid GA, and add 20 μ l Proteinase Ks (20mg/ml), 56 ℃ of water-bath effect 2h;
1.2 add 200 μ l damping fluid GB and place 70 ℃ of water-bath effect 10min, briefly centrifugal;
1.3 add 200 μ l dehydrated alcohols, brief centrifugal afterwards supernatant being transferred among the centrifugal post CB3, the centrifugal 1min of 12000r/min discards the collection liquid in pipe;
1.4 add 500 μ l protein liquid removals toward centrifugal post CB3, the centrifugal 1min of 12000r/min discards the collection liquid in pipe;
1.5 add 700 μ l rinsing liquid PW toward centrifugal post CB3, the centrifugal 1min of 12000r/min discards the collection liquid in pipe;
Wash once more 1.6 add 500 μ l rinsing liquid PW toward centrifugal post CB3, discard the collection liquid in pipe, the centrifugal 2min of blank pipe 12000r/min puts room temperature 2～5min and dries;
1.7 with 20 μ lTE dissolving, the centrifugal 2min of 12000r/min is collected in the new EP pipe at last.
2.PCR primer and probe:
Query analysis NCBI goes up listed Trichinella spiralis nucleotide sequence, chooses the target sequence of Trichinella spiralis plastosome Ls-rRNA gene as pcr amplification, and it is right that following primer has been synthesized in design:
F：5′-GGTTGCAGTCTTAAAGAGAATCC-3′
R：5′-CGAGAATGAAAGGAGTAAAGAAAG-3′
Trichina(Trichinella spiralis), local hair shape nematode, pig Trichinella spiralis U.S. isolated species, pig Trichinella spiralis Heilungkiang isolated species and dog Trichinella spiralis Heilungkiang isolated species genomic dna are increased, the sequence of amplification is compared, and choosing LS-rRNA gene conserved regions design, to have synthesized following primer right:
P1：5’-TGCAGTCTTAAAGAGAATCCAACCT-3’，
P2：5’-CAAGTTTCGTCTGTTCGACGATA-3’，
The expection amplified production is approximately 67bp.
Synthesized a TaqMAN MGB probe according to the sequences Design between the primer:
Probe:5 '-Fam-TTGCGACGGTTTAAA-NFQ-MGB-3 ', at its 5 ' end mark fluorescent dyestuff Fam, 3 ' holds the then non-fluorescent quenching group of mark (Non-Fluorescent Quencher), also connects MGB (Minor GrooveBinder) modification group simultaneously, to improve the Tm value.
3. the preparation of positive quality control standard substance:
Conventional PCR product is connected with cloning vector pMD-18-T vector, is transformed into e. coli tg1, and preserve positive reorganization bacterium.Prepare plasmid and carry out quantitative with reference to MiniBEST Plasmid Purification Kit specification sheets.
3.1 colibacillary cultivation.Select the antibiotic liquid nutrient medium of containing of single colony inoculation to 1～4ml 37 ℃ of incubated overnight from plate culture medium;
3.2 get the incubated overnight bacterium liquid of 1～4ml, the centrifugal 2min of 12000r/min abandons supernatant;
3.3 bacterial precipitation fully suspends with the Solution I (containing RNase A1) of 250 μ l;
Spin upside down mixing 5～6 times lightly 3.4 add the Solution II of 250 μ l, make the abundant cracking of thalline, form clear solution;
3.5 add the Solution III of 4 ℃ of precoolings of 400 μ l, spin upside down gently and mix 5～6 times, until forming consolidation aggegation piece, room temperature leaves standstill 2min then;
3.6 the centrifugal 10min of room temperature 12000r/min gets supernatant;
3.7 the Spin Column in the test kit is placed on the Collection Tube;
3.8 the supernatant liquor of aforesaid operations 6 is transferred among the Spin Column, and the centrifugal 1min of 12000r/min abandons filtrate;
3.9 the RinseA of 500 μ l is added among the Spin Column, and the centrifugal 30s of 12000r/min abandons filtrate.
3.10 the Rinse B of 700 μ l is added among the Spin Column, and the centrifugal 30s of 12000r/min abandons filtrate.
3.11 repetitive operation step 10.
3.12 Spin Column is placed on the centrifuge tube of new 1.5ml, add sterile purified water or the Elution Buffer of 60 μ l in the centre of Spin Column film, room temperature leaves standstill 1min.
3.13 the centrifugal 1min eluted dna of 12000r/min.
Draw 50 μ l and carry out quantitatively after extracting plasmid, concrete grammar is as follows: get 10 μ l plasmids and be diluted to 500 μ l, survey OD
260, calculate the quality of plasmid then, calculate volumetric molar concentration again, multiply by Avogadro constant (6.02 * 10 at last
23), be the copy number of unit volume.Calculation formula is:
Positive plasmid copy number (copies/ μ l)=(OD
260* 50 * 10
-9* extension rate * 6.02 * 10
23)/(660 * base number)
In the formula: 50 representatives use the cuvette of 1cm at OD
260Be that 1 o'clock corresponding double-stranded DNA concentration is 50 μ g/ml;
660 represent the molecular weight of average each base pair of double-stranded DNA.
Make the positive quality control standard substance of different concns gradient with 5 or 10 multiple proportions serial dilution ,-20 ℃ of preservations are standby.
4. the foundation of reaction system and optimization:
Positive quality control standard substance to the different concns gradient carry out trial test, choose the concentration (4.12 * 10 of Ct about 20
5Template during copies/ μ l) as system optimization.
4.1 rTaq enzyme single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system, the consumption of rTaq enzyme at 0.25～2 μ l, is increased progressively with per 0.25 μ l, by the comparative analysis of test-results, the consumption of determining best rTaq enzyme is 0.3 μ l.
4.2 dNTP single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system, the dNTP amount ranges is located at each reaction 0.5～2.5 μ l, increases progressively, through the dNTP consumption of the selected 1.5 μ l of revision test repeatedly as each reaction in the test kit with per 0.25 μ l.
4.3 10 * PCR Buffer single-factor concentration gradient is optimized
Through 10 * PCR Buffer test-results of using different concns relatively, selected 1 * as the PCR damping fluid final concentration of each reaction in the test kit.
4.4 probe single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system the probe amount ranges is located at each reaction 0.10～0.50 μ l, increases progressively, revision test is compared analysis, determine that the best probe consumption of each reaction is 0.30 μ l in the test kit with per 0.05 μ l.
4.5 the optimization of primer concentration
Under the situation that other conditions are identical in reaction system, upstream and downstream primer consumption is located at each reaction 0.10～0.90 μ l respectively, increase progressively with per 0.10 μ l, matrix analysis determines that the best primer consumption of each reaction is that the upstream and downstream primer is 0.50 μ l in the test kit.
4.6 Mg
2+The single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system, with MgCl
2Consumption increase progressively with per 0.4 μ l from 0～6.0 μ l, through the MgCl of the selected 2.4 μ l of revision test repeatedly as each reaction in the test kit
2Consumption.
4.7 determining of loop parameter
Sex change time, annealing and elongating temperature, time and cycle index are carried out suitable adjusting, and it is the shortest to choose detection time, and the best loop parameter of amplification curve is as the temperature and time parameter of PCR, i.e. 95 ℃ of pre-sex change 2min; 95 ℃ of sex change 10s, 30s is extended in 60 ℃ of annealing, and 40cycles collects fluorescent signal in the time of 60 ℃.
Utilize above-mentioned primer and probe to carry out the foundation of reaction system, determine that at last the Trichinella spiralis real-time fluorescence PCR detection architecture that adopts is 25 μ l, required each component and corresponding consumption are as follows:
Aseptic double-distilled water 12 μ l
10 times of reaction buffers (10 *), 2.5 μ l
MgCl
2(25mM) 2.4μl
Thymus nucleic acid (dNTP) is 1.5 μ l (2.5mM)
Primer 1 (10 μ M) 0.5 μ l
Primer 2 (10 μ M) 0.5 μ l
TaqMAN?MGB?Probes(10μM) 0.3μl
Taq archaeal dna polymerase (5U/ μ l) 0.3 μ l
Reaction conditions is: 95 ℃ of pre-sex change 2min; 95 ℃ of sex change 10s, 30s is extended in 60 ℃ of annealing, and 40cycles collects fluorescent signal in the time of 60 ℃.
5. specificity test
With the universal real time fluorescent PCR detection method of trichinella of setting up to trichina(Trichinella spiralis), local hair shape nematode, pig Trichinella spiralis U.S. isolated species, pig Trichinella spiralis Heilungkiang isolated species, the genomic dna of dog Trichinella spiralis Heilungkiang isolated species genomic dna and ascaris suum, trichuris suis, pig hookworm, ascaris alata, haemonchus contortus, toxoplasma gondii, healthy pork, dog meats, beef, mouse muscle and water contrast detect, to probe into the specificity that this method detects.
6. susceptibility test
The positive quality control standard substance of different concns gradient with preparation are pcr template, increase on the fluorescent PCR instrument by above-mentioned PCR reaction system and response procedures, the machine analysis draws the Ct value of different copy numbers as calculated, logarithm according to Ct value and respective copies number is made typical curve, calculate amplification efficiency, E=10
-1/K-1, wherein the K value is the slope of typical curve.
Under anatomical lens, draw the wall scroll Trichinella spiralis to clean 1.5ml EP pipe with 10 μ l pipettors, extract the genomic dna of wall scroll Trichinella spiralis with 1 described method, multiple proportions serial dilution with 10 is respectively got 5 μ l as template, make each the reaction in contained genome be equivalent to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5The bar Trichinella spiralis is established three and repeats to detect, and investigates the absolute sensitivity that this method detects.
7. replica test
Detect simultaneously 7.1 same positive quality control standard substance are established 20 multiple pipes, calculate the variation coefficient of Ct value, the repeatability of investigation detection method and stable.
7.2 the positive quality control standard substance of the different concns gradient that detects in the susceptibility test, heavily inspection behind-20 ℃ of preservation 30d, the Ct value of twice detection compares test of hypothesis, observes whether to have significant difference, investigates the stability that the inventive method detects.
Claims (7)
1, a kind of universal real time fluorescent PCR detection method of trichinella, it is characterized in that choosing the target sequence of each Trichinella spiralis isolated species plastosome LS-rRNA gene conserved regions sequence as pcr amplification, set synthetic specific primer and TaqMAN MGB probe, Trichinella spiralis is carried out qualitative detection.
2, a kind of universal real time fluorescent PCR detection method of trichinella according to claim 1, it is characterized in that setting synthetic following primer to TaqMAN MGB probe:
P1:5’-TGCAGTCTTAAAGAGAATCCAACCT-3’，
P2:5’-CAAGTTTCGTCTGTTCGACGATA-3’，
Probe:5’-Fam-TTGCGACGGTTTAAA-NFQ-MGB-3’。
3, a kind of universal real time fluorescent PCR detection method of trichinella according to claim 2, it is characterized in that Trichinella spiralis comprises trichina(Trichinella spiralis) (Trichinella spiralis, T1), local hair shape nematode (Trichinella nativa, T2), pig Trichinella spiralis Heilungkiang isolated species (Trichinella swine isolate from Heilongjiang), dog Trichinella spiralis Heilungkiang isolated species (Trichinella dog isolate from Heilongjiang), pig Trichinella spiralis U.S. isolated species (Trichinella swine isolatefrom USA) detects.
4, a kind of universal real time fluorescent PCR detection method of trichinella according to claim 3 is characterized in that described pcr amplification reaction reaction system is:
Aseptic double-distilled water 12 μ l
10 times of reaction buffer 10 * 2.5 μ l
MgCl
2?25mM 2.4μl
Thymus nucleic acid dNTP 2.5mM 1.5 μ l
Primer 110 μ M 0.5 μ l
Primer 2 10 μ M 0.5 μ l
TaqMAN?MGB?Probes?10μM 0.3μl
Template DNA 5 μ l
Taq archaeal dna polymerase 5U/ μ l 0.3 μ l
Reaction conditions is: 95 ℃ of pre-sex change 2min; 95 ℃ of sex change 10s, 30s is extended in 60 ℃ of annealing, and 40cycles collects fluorescent signal in the time of 60 ℃.
5, a kind of universal real time fluorescent PCR detection method of trichinella according to claim 4 is characterized in that:
1) preparation of .PCR template:
1.1 get the 0.1g detected sample, add 200 μ l damping fluid GA, and add 20 μ l Proteinase Ks (20mg/ml), 56 ℃ of water-bath effect 2h;
1.2 add 200 μ l damping fluid GB and place 70 ℃ of water-bath effect 10min, briefly centrifugal;
1.3 add 200 μ l dehydrated alcohols, brief centrifugal afterwards supernatant being transferred among the centrifugal post CB3, the centrifugal lmin of 12000r/min discards the collection liquid in pipe;
1.4 add 500 μ l protein liquid removals toward centrifugal post CB3, the centrifugal 1min of 12000r/min discards the collection liquid in pipe;
1.5 add 700 μ l rinsing liquid PW toward centrifugal post CB3, the centrifugal 1min of 12000r/min discards the collection liquid in pipe;
Wash once more 1.6 add 500 μ l rinsing liquid PW toward centrifugal post CB3, discard the collection liquid in pipe, the centrifugal 2min of blank pipe 12000r/min puts room temperature 2～5min and dries;
1.7 with 20 μ lTE dissolving, the centrifugal 2min of 12000r/min is collected in the new EP pipe at last;
2) .PCR primer and probe: choose the target sequence of the big subunit rRNA of Trichinella spiralis plastosome gene as pcr amplification, it is right to set synthetic following primer:
F:5′-GGTTGCAGTCTTAAAGAGAATCC-3′
R:5′-CGAGAATGAAAGGAGTAAAGAAAG-3′
Trichina(Trichinella spiralis), local hair shape nematode, pig Trichinella spiralis U.S. isolated species, pig Trichinella spiralis Heilungkiang isolated species and dog Trichinella spiralis Heilungkiang isolated species genomic dna are increased, the sequence of amplification is compared, and choosing LS-rRNA gene conserved regions design, to have synthesized following primer right:
P1:5’-TGCAGTCTTAAAGAGAATCCAACCT-3’，
P2:5’-CAAGTTTCGTCTGTTCGACGATA-3’，
The expection amplified production is approximately 67bp;
Synthesized a TaqMAN MGB probe according to the sequences Design between the primer:
Probe:5 '-Fam-TTGCGACGGTTTAAA-NFQ-MGB-3 ', at its 5 ' end mark fluorescent dyestuff Fam, 3 ' holds the then non-fluorescent quenching group of mark, also connects the MGB modification group simultaneously;
3). the preparation of positive quality control standard substance:
Conventional PCR product is connected with cloning vector pMD-18-T vector, is transformed into e. coli tg1, and preserve positive reorganization bacterium, the preparation plasmid also carries out quantitatively;
3.1 colibacillary cultivation is selected the antibiotic liquid nutrient medium of containing of single colony inoculation to 1～4ml from plate culture medium, 37 ℃ of incubated overnight;
3.2 get the incubated overnight bacterium liquid of 1～4ml, the centrifugal 2min of 12000r/min abandons supernatant;
3.3 contain the RNase A1 bacterial precipitation that fully suspends with the Solution I of 250 μ l;
Spin upside down mixing 5～6 times lightly 3.4 add the Solution II of 250 μ l, make the abundant cracking of thalline, form clear solution;
3.5 add the Solution III of 4 ℃ of precoolings of 400 μ l, spin upside down gently and mix 5～6 times, until forming consolidation aggegation piece, room temperature leaves standstill 2min then;
3.6 the centrifugal 10min of room temperature 12000r/min gets supernatant;
3.7 the Spin Column in the test kit is placed on the Collection Tube;
3.8 the supernatant liquor of aforesaid operations 6 is transferred among the Spin Column, and the centrifugal 1min of 12000r/min abandons filtrate;
3.9 the RinseA of 500 μ l is added among the Spin Column, and the centrifugal 30s of 12000r/min abandons filtrate;
3.10 the Rinse B of 700 μ l is added among the Spin Column, and the centrifugal 30s of 12000r/min abandons filtrate;
3.11 repetitive operation step 10;
3.12 Spin Column is placed on the centrifuge tube of new 1.5ml, add sterile purified water or the Elution Buffer of 60 μ l in the centre of Spin Column film, room temperature leaves standstill 1min;
3.13 the centrifugal 1min eluted dna of 12000r/min;
Draw 50 μ l and carry out quantitatively after extracting plasmid, make the positive quality control standard substance of different concns gradient with 5 or 10 multiple proportions serial dilution ,-20 ℃ of preservations are standby;
4). the foundation of reaction system and optimization:
Positive quality control standard substance to the different concns gradient carry out trial test, choose the concentration 4.12 * 10 of Ct about 20
5The template of copies/ μ l during as system optimization.
6, a kind of universal real time fluorescent PCR detection method of trichinella according to claim 5 is characterized in that: draw 50 μ l and carry out quantitative concrete grammar and be after extracting plasmid: get 10 μ l plasmids and be diluted to 500 μ l, survey OD
260, calculate the quality of plasmid then, calculate volumetric molar concentration again, multiply by Avogadro constant 6.02 * 10 at last
23, being the copy number of unit volume, calculation formula is:
Positive plasmid copy number copies/ μ l=(OD
260* 50 * 10
-9* extension rate * 6.02 * 10
23)/(660 * base number)
In the formula: 50 representatives use the cuvette of 1cm at OD
260Be that 1 o'clock corresponding double-stranded DNA concentration is 50 μ g/ml;
660 represent the molecular weight of average each base pair of double-stranded DNA.
7, a kind of universal real time fluorescent PCR detection method of trichinella according to claim 6 is characterized in that the foundation of reaction system and optimization step comprise:
1.1 rTaq enzyme single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system, the consumption of rTaq enzyme at 0.25～2 μ l, is increased progressively with per 0.25 μ l, by the comparative analysis of test-results, the consumption of determining best rTaq enzyme is 0.3 μ l;
1.2 dNTP single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system, the dNTP amount ranges is located at each reaction 0.5～2.5 μ l, increases progressively, through the dNTP consumption of the selected 1.5 μ l of revision test repeatedly as each reaction in the test kit with per 0.25 μ l;
1.3 10 * PCR Buffer single-factor concentration gradient is optimized
Through 10 * PCR Buffer test-results of using different concns relatively, selected 1 * as the PCR damping fluid final concentration of each reaction in the test kit;
1.4 probe single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system the probe amount ranges is located at each reaction 0.10～0.50 μ l, increases progressively, revision test is compared analysis, determine that the best probe consumption of each reaction is 0.30 μ l in the test kit with per 0.05 μ l;
1.5 the optimization of primer concentration
Under the situation that other conditions are identical in reaction system, upstream and downstream primer consumption is located at each reaction 0.10～0.90 μ l respectively, increase progressively with per 0.10 μ l, matrix analysis determines that the best primer consumption of each reaction is that the upstream and downstream primer is 0.50 μ l in the test kit;
1.6 Mg
2+The single-factor concentration gradient is optimized
Under the situation that other conditions are identical in reaction system, with MgCl
2Consumption increase progressively with per 0.4 μ l from 0～6.0 μ l, through the MgCl of the selected 2.4 μ l of revision test repeatedly as each reaction in the test kit
2Consumption;
1.7 determining of loop parameter
Sex change time, annealing and elongating temperature, time and cycle index are carried out suitable regulating part, and it is the shortest to choose detection time, and the best loop parameter of amplification curve is as the temperature and time parameter of PCR, i.e. 95 ℃ of pre-sex change 2min; 95 ℃ of sex change 10s, 30s is extended in 60 ℃ of annealing, and 40cycles collects fluorescent signal in the time of 60 ℃.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519682B (en) * | 2009-03-26 | 2012-01-11 | 张舒亚 | Method for detecting cat source components in food and fodder and kit |
| CN102424841A (en) * | 2011-12-21 | 2012-04-25 | 中国人民解放军疾病预防控制所 | LAMP detection method for trichinella spiralises, special primers and kit thereof |
| CN102839218A (en) * | 2012-09-19 | 2012-12-26 | 东北农业大学 | Method for detecting trichina and cysticercus cellulosae in food |
| CN105274237A (en) * | 2015-11-17 | 2016-01-27 | 南京农业大学 | Method and primer combination for quickly detecting trichinella spiralis on basis of LAMP (loop-mediated isothermal amplification) technologies |
| CN106053403A (en) * | 2016-04-29 | 2016-10-26 | 浙江大学 | A protein quantitative detection method |
| CN112410437A (en) * | 2020-12-07 | 2021-02-26 | 沈阳农业大学 | Real-time fluorescent quantitative PCR method for detecting sheep meat product trichina |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100487138C (en) * | 2006-08-10 | 2009-05-13 | 中国检验检疫科学研究院动植物检疫研究所 | Roundworm egg detecting real-time fluorescence PCR primer and probe |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101519682B (en) * | 2009-03-26 | 2012-01-11 | 张舒亚 | Method for detecting cat source components in food and fodder and kit |
| CN102424841A (en) * | 2011-12-21 | 2012-04-25 | 中国人民解放军疾病预防控制所 | LAMP detection method for trichinella spiralises, special primers and kit thereof |
| CN102839218A (en) * | 2012-09-19 | 2012-12-26 | 东北农业大学 | Method for detecting trichina and cysticercus cellulosae in food |
| CN105274237A (en) * | 2015-11-17 | 2016-01-27 | 南京农业大学 | Method and primer combination for quickly detecting trichinella spiralis on basis of LAMP (loop-mediated isothermal amplification) technologies |
| CN105274237B (en) * | 2015-11-17 | 2018-03-06 | 南京农业大学 | A kind of trichina quick determination method and Primer composition based on LAMP technology |
| CN106053403A (en) * | 2016-04-29 | 2016-10-26 | 浙江大学 | A protein quantitative detection method |
| CN112410437A (en) * | 2020-12-07 | 2021-02-26 | 沈阳农业大学 | Real-time fluorescent quantitative PCR method for detecting sheep meat product trichina |
| CN112410437B (en) * | 2020-12-07 | 2024-01-26 | 沈阳农业大学 | Real-time fluorescence quantitative PCR method for detecting trichina in sheep meat product |
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