CN112410437B - Real-time fluorescence quantitative PCR method for detecting trichina in sheep meat product - Google Patents
Real-time fluorescence quantitative PCR method for detecting trichina in sheep meat product Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a real-time fluorescence quantitative PCR method for detecting trichina in sheep meat products, which comprises the following steps: designing a primer according to the Scfld4 gene of the trichina iss195 strain chromosome; diluting the extracted trichina genes by a multiple ratio concentration, and respectively carrying out real-time fluorescence quantitative PCR detection on the trichina full-length gene samples diluted by the multiple ratio concentration by using the primers; establishing a standard curve by taking CT values of the trichina genes diluted by the multiple ratio concentration as an ordinate and taking logarithm of the corresponding DNA concentration as an abscissa; and detecting a sample to be detected by using real-time fluorescence quantitative PCR, and calculating the corresponding concentration of the trichina genes in the sample to be detected according to the CT value of the sample to be detected and the standard curve. The invention designs the specific primer by using the fluorescent dye method, establishes the method of real-time fluorescent quantitative PCR, and has the characteristics of strong sensitivity, good stability, high flux and the like.
Description
Technical Field
The invention relates to the technical field of PCR detection, in particular to a real-time fluorescent quantitative PCR method for detecting trichina in sheep meat products.
Background
Trichinosis is a serious parasitic disease of human and animals, which causes great economic loss for animal husbandry production and also forms a great threat to human health. Humans or animals (pigs, herbivores, etc.) are mainly ill by feeding on meat contaminated with trichina muscle larvae by mistake.
Currently, with respect to the diagnosis of trichina, the international veterinary agency (OIE) accepted methods are the tableting microscopy and digestion. At present, two methods are used in China, however, the two methods have certain defects in detection, and the microscopic examination method is time-consuming and labor-consuming and has lower sensitivity. Although the digestion method can improve the detection rate of the trichina to a certain extent, the method has the advantages that the detection rate of the trichina can be detected only by more than 3 trichina contained in each gram of meat, and the method is extremely tedious in operation and consumes a great amount of manpower and material resources. From the point of biosafety, missed meat products caused by microscopy and digestion still pose a threat to human health (ingestion of about 75 worms can cause infection in humans). A great deal of research is carried out on methods for detecting the trichina in pigs by students at home and abroad, such as common PCR, PCR chips and the like are applied to detect the trichina samples, but the methods are not suitable for detecting large-scale trichina samples due to low sensitivity and high price.
Disclosure of Invention
Therefore, the invention provides a real-time fluorescence quantitative PCR method for detecting trichina in a sheep meat product, which aims to solve the problems of low sensitivity and high price in the prior art and is not suitable for detecting mass trichina samples.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a real-time fluorescence quantitative PCR method for detecting trichina in a sheep meat product, which comprises the following steps:
designing a primer according to the Scfld4 gene of the trichina iss195 strain chromosome;
diluting the extracted trichina genes by a multiple ratio concentration, and respectively carrying out real-time fluorescence quantitative PCR detection on the trichina full-length gene samples diluted by the multiple ratio concentration by using the primers;
establishing a standard curve by taking CT values of the trichina genes diluted by the multiple ratio concentration as an ordinate and taking logarithm of the corresponding DNA concentration as an abscissa;
and detecting a sample to be detected by using real-time fluorescence quantitative PCR, and calculating the concentration corresponding to the trichina gene in the sample to be detected according to the CT value of the sample to be detected and the standard curve.
In one embodiment of the present invention, the amplification primers of the Scfld4 gene are:
an upstream primer: 5'-TTCTTAGTGCTTTCTGGGTCATCTT-3';
a downstream primer: 5'-CGCTTTTCCACTTACCATTCTGT-3'.
In one embodiment of the present invention, the diluted concentrations of the trichina genes are respectively: 41.10 ng/. Mu.L, 8.22 ng/. Mu.L, 1.644 ng/. Mu.L, 0.3288 ng/. Mu.L, 0.06576 ng/. Mu.L, 0.013152 ng/. Mu.L, 0.002630 ng/. Mu.L.
In one embodiment of the invention, the trichina is a T1-type trichina.
In one embodiment of the present invention, the PCR reaction system is: 1X MonAmpTM ChemoHS qPCR Mix. Mu.L; 0.2. Mu.M forward primer 0.4. Mu.L; 0.2. Mu.M reverse primer 0.4. Mu.L; 1X Low Rox Dye or High Rox Dye 0.2.2. Mu.L; 9 mu L of DNA template; nuclear-Free Water To 20. Mu.L.
In one embodiment of the invention, the PCR reaction conditions are: 95℃for 10min,95℃for 10s,60℃for 10s,72℃for 30s,40cycles.
In one embodiment of the present invention, the standard curve is: y= -3.28x+23.531, where 18.ltoreq.y.ltoreq.35.
The invention has the following advantages:
the invention designs a specific primer by using a fluorescent dye method, establishes a real-time fluorescent quantitative PCR method, has the characteristics of strong sensitivity, good stability, high flux and the like, and firstly uses a novel gene to specially aim at the molecular diagnosis method established by the detection method of T1 trichina in sheep meat products.
The real-time fluorescence quantitative PCR detection method not only realizes the leap of PCR from qualitative to quantitative, but also has high sensitivity and low price, and can detect a single sample and a plurality of samples.
Drawings
FIG. 1 is a graph showing the real-time fluorescent quantitative PCR amplification of 100 DNA fragments of the trichina after extraction and purification, wherein the DNA fragments of the trichina are 5 1 、5 2 、5 3 、5 4 、5 5 、5 6 、5 7 Dilution is carried out in which Y 1 Corresponding to 5 1 Dilution, Y 2 Corresponding to 5 2 Dilution, Y 3 Corresponding to 5 3 Dilution, Y 4 Corresponding to 5 4 Dilution, Y 5 Corresponding to 5 5 Dilution, Y 6 Corresponding to 5 6 Dilution, Y 7 Corresponding to 5 7 Diluting;
FIG. 2 is a graph of a real-time fluorescent quantitative PCR standard provided by the present invention, in which the DNA of the trichina body is obtained at a ratio of 5 1 、5 2 、5 3 、5 4 、5 5 、5 6 、5 7 Diluted with 5 7 The diluted DNA detection concentration is 0.002630 ng/. Mu.L, the CT value of the real-time fluorescence quantitative PCR detection of the trichina gene diluted by the double-ratio concentration is taken as an ordinate, and the logarithm of the corresponding DNA concentration is taken as an abscissa;
FIG. 3 is a real-time fluorescent quantitative PCR amplification chart of the Bursaphelenchus xylophilus provided by the invention, wherein 1 represents a positive control, 2 represents the Bursaphelenchus xylophilus, and 3 represents a blank control;
FIG. 4 is a real-time fluorescence quantitative PCR amplification chart of the sheep supreme nematode provided by the invention, wherein 1 represents a positive control, 2 represents the sheep supreme nematode, and 3 represents a blank control;
FIG. 5 is a real-time fluorescent quantitative PCR amplification chart of the aphelenchus xylophilus provided by the invention, wherein 1 represents a positive control, 2 represents the aphelenchus xylophilus, and 3 represents a blank control;
FIG. 6 is a real-time fluorescent quantitative PCR amplification chart of the strongyloid provided by the invention, wherein 1 represents a positive control, 2 represents the strongyloid, and 3 represents a blank control;
FIG. 7 is a graph showing the results of the real-time fluorescence quantitative PCR detection of different numbers of the artemia salina in the same quality tissue, wherein 1 represents 40, 2 represents 20, 3 represents 10, 4 represents 5, 5 represents 1, 6 represents a blank control, and 7 represents a negative control;
FIG. 8 is a graph showing the results of a sensitivity verification of a real-time fluorescent quantitative PCR for detecting different tissue qualities, wherein 1 represents 1mg,2 represents 5mg,3 represents 10mg,4 represents 20mg,5 represents a blank control, and 6 represents a negative control;
FIG. 9 is a view of a microscope provided by the invention for detecting trichina in the sheep's attack diaphragm;
FIG. 10 is a graph of real-time fluorescent quantitative PCR results of the diaphragm muscle of an offensive sheep provided by the invention, wherein 1 represents an offensive group, 2 represents a blank control, and 3 represents a negative control;
FIG. 11 is a graph of the results of the detection of suspected tissues of 73 sheep, wherein 1 represents positive control, 2 represents No. 8, 3 represents No. 14, 4 represents blank control, and 5 represents negative control;
fig. 12 is a graph of the results of the detection of 73 sheep suspected tissues provided by the invention, wherein 1 represents a positive control, 2 represents No. 32, 3 represents No. 37, 4 represents No. 27, 5 represents a blank control, and 6 represents a negative control.
Detailed Description
Example 1 Standard Curve establishment of real-time fluorescence quantitative PCR for detection of Trichinella spiralis in a sheep meat product
1. Design real-time fluorescent quantitative primer
Screening of detection genes of trichina in sheep meat products by real-time fluorescent quantitative PCR method
Gene number (Gene number: tsp-00874) of the Scfld4 gene unknown to the chromosome of the trichina iss195 strain was selected from Global Gene Expression Analysis of theZoonotic Parasite Trichinella spiralis Revealed NovelGenes in Host ParasiteInteraction published in the previous laboratory study: 10904414 the full-length gene of this gene was then found in GenBank gene pool in NCBI, which was up-regulated during muscle larval stage, and was found to be conserved by BLAST analysis.
Primer design of real-time fluorescence quantitative PCR method for trichina in sheep meat products,
an upstream primer: 5'-TTCTTAGTGCTTTCTGGGTCATCTT-3';
a downstream primer: 5'-CGCTTTTCCACTTACCATTCTGT-3'.
2. Extraction of sample tissue DNA of Trichinella spiralis
The preparation was carried out with reference to the instructions of the general genomic DNA extraction kit for stearypure, and the appropriate amount of sample was taken:
1. adding 500 mu L buffer Ls-2, 20 mu L protease k and 10 mu L RNase A (10 mg/mL), and heating in a water bath at 56 ℃ until the insect body is completely cracked (about 2-3 h);
2. the sample is fully cracked by untimely inversion, 200 mu L Buffer BS-2 and 200 mu L100% ethanol are added into the cracking liquid, and the mixture is fully sucked and uniformly mixed;
3. transferring the solution into a m niversal DNA Mini Column, standing at room temperature for 1min, centrifuging at 12000rpm for 1min at room temperature, and discarding the filtrate;
4. 500. Mu.L of Buffer WA is added to the Mini Column, the mixture is centrifuged at 12000rpm for 1 minute at room temperature, and the filtrate is discarded;
5. 750 mu L Buffer WB is added into Mini Column, the mixture is centrifuged for 1 minute at 12000rmp at room temperature, and the filtrate is discarded;
6. repeating the step 3 once;
7. mini Column was mounted on a new 2mL Collection Tube, centrifuged at 12000rpm for 2 minutes at room temperature;
8. mini Column was placed in a new 1.5mL centrifuge tube, 45. Mu.L of sterilized water was added at the center of the Mini Column membrane, left standing at room temperature for 5 minutes, and then centrifuged at 12000rpm for 2 minutes, and collected in a new EP tube.
3. Standard Curve establishment
Taking 100 purified clean worms, extracting trichina full-length gene DNA, diluting positive standard substances by 5 times to obtain 7 points, and naming the points as Y n (n＝1～7)。
The reaction system is as follows: 1X MonAmpTM ChemoHS qPCR Mix. Mu.L, 0.2. Mu.M forward primer (10. Mu.M) a 0.4. Mu.L, 0.2. Mu.M reverse primer (10. Mu.M) a 0.4. Mu.L, 1X Low Rox Dye (100X) or High Rox Dye (100) b 0.2. Mu. L, DNA template 9. Mu. L, nuclease-Free Water To 20. Mu.L.
The reaction conditions were 95℃for 10min,95℃for 10s,60℃for 10s,72℃for 30s,40cycles.
Positive standard substances are serially diluted for 7 times by 5 times and respectively marked as Y 1 ～Y 7 ，Y 1 The concentration of DNA extracted from the insect bodies was 100. Y is Y 1 ～Y 7 The DNA concentration of (2) is: 41.10 ng/. Mu.L, 8.22 ng/. Mu.L, 1.644 ng/. Mu.L, 0.3288 ng/. Mu.L, 0.06576 ng/. Mu.L, 0.013152 ng/. Mu.L, 0.002630 ng/. Mu.L. Y is prepared by established real-time fluorescence quantitative PCR method 1 ～Y 7 Detecting templates with different concentration gradients, wherein amplification curves of templates show good parallel relation in an index range, as shown in fig. 1, CT values of trichina gene real-time fluorescence quantitative PCR detection with diluted multiple ratio concentration are taken as ordinate, logarithm of corresponding DNA concentration is taken as abscissa, a standard curve is established, as shown in fig. 2, the amplification efficiency is 101.7%, the amplification curves show good linear relation, and the standard curve is: y= -3.28x+23.531, where Y represents CT value for fluorescent quantitative PCR detection, range of CT: CT is more than or equal to 18 and less than or equal to 35; x represents the logarithm of DNA concentration, DNA concentration detectable range: 0.0003186 ng/. Mu.L the concentration of DNA is less than or equal to 48.56 ng/. Mu.L; correlation coefficient R 2 ＝0.9993。
The amount of DNA in the detection system is equivalent to the DNA mass of 100, 20, 4, 0.8, 0.16, 0.132 and 0.0064 trichina, and the test detects Y 7 The DNA concentration of (C) was 0.002630 ng/. Mu.L, which corresponds to detection of one thousandth of a insect.
Test example 1, specificity test
The established real-time fluorescence PCR detection method is used for detecting the full-length gene DNA of the trichina, the esophageal mouth nematodes, the blood-twisting nematodes and the sheep supreme nematodes, the DNA group extracted from the trichina is set as a positive sample group, and at least 3 parallel double tubes and 3 blank controls are set. The CT average value of the positive sample DNA detection is 31.959, the DNA of the esophageal mouth nematodes, the DNA of the supreme sheep mouth nematodes, the DNA of the pralidoxime blood nematodes, the DNA detection of the trichostrongylus, and the blank control are not peaked, the detection result is negative, no cross reaction exists, the specificity is good, the DNA detection result of the esophageal mouth nematodes is shown in figure 5, the DNA detection result of the pralidoxime sheep mouth nematodes is shown in figure 4, the DNA detection result of the pralidoxime blood nematodes is shown in figure 6, and the DNA detection result of the trichostrongylus is shown in figure 3.
In the specificity test, the DNA of the esophageal eelworms, the DNA of the supreme ovine eelworms, the DNA of the haemonchus contortus and the DNA of the trichodesmus, and a blank control do not peak, the detection result is negative, no cross reaction exists, and the specificity is good.
Test example 2, sensitivity test
1, 5, 10, 20 and 40 insects were added to 5mg sheep diaphragmatic muscle, respectively, and DNA was extracted and detected as shown in FIG. 7. 1mg, 5mg, 10mg and 20mg of sheep diaphragmatic tissue are respectively added into 1 insect, DNA is extracted for detection, and the results show that 100% of the detection can be achieved, as shown in figure 8.
The detection results are that the muscle tissues with the same muscle tissue, different insect numbers and different masses are smaller, the detection rate is higher, the sensitivity is higher, and the detection results are that the detection rate is higher and the sensitivity is higher.
The minimum detection amount is 20mg of sheep diaphragmatic tissue DNA detection samples containing 1 trichina in different tissues or the fluorescence signal can be detected in 5mg of sheep diaphragmatic muscle added with 1 trichina DNA detection sample, which indicates that the detection method has high sensitivity.
Test example 3 detection of Positive samples
And (one) tissue trichina extraction test:
(1) 1 Kunming mouse after 35 days of feeding by gastric lavage and insect killing is taken;
(2) After neck removal and sacrifice, the mixture was soaked in 75% alcohol for 1min and then in PBS for 1min.
(3) The limbs, tail, viscera and adipose tissues of the mice were removed, leaving only the carcass.
(4) Diaphragmatic muscle is taken for tabletting microscopy, and trichina cysts and muscle larvae are found under a microscope (inverted microscope).
(5) The carcass is weighed.
(6) Artificial digest (1% hcl,1% pepsin) was prepared: 6mL of HCL solution is dissolved in 400mL of distilled water or ultra-pure water, and then 6g of pepsin is added to fix the volume to 600mL (which is used in the prior art).
(7) The carcass and the checked diaphragm are sheared into small sand grains, and the small sand grains are split into two conical flasks of 250 mL.
(8) 120mL of artificial digest (typically 15 times the carcass volume) was added to each.
(9) The flask mouth was closed, 220rpm,37℃for 3h.
(10) The flask was removed, the meat residue was filtered through a 60 mesh screen, filtered into a 500mL measuring cup, and then added with 37℃physiological saline to 500mL, and neutralized for digestion.
(11) Standing in a constant temperature incubator at 37deg.C for 30min.
(12) The standing solution was removed, 400mL of the upper layer liquid was removed, and 100mL of the lower layer suspension was left.
(13) The suspension was poured into a 250mL measuring cup, physiological saline was added to 250mL, and the mixture was left to stand in a 37℃incubator for 35min.
(14) Taking out the standing liquid, removing 200mL of the upper liquid, leaving 50mL of the lower suspension, pouring the suspension into a 100mL measuring cup, adding physiological saline to 100mL, and standing for 30min at 37 ℃ in an incubator.
(15) Taking out the standing liquid, removing 80mL of upper liquid, keeping 20mL of lower suspension, standing at room temperature for 15min, removing supernatant, mixing, adding into a 1.5mL centrifuge tube, microscopic examination activity, counting, and preserving at about 6000 pieces at 4 ℃.
(II) test for detecting Positive samples
About 6000 trichina muscle larvae were extracted from the Kunming mice in a laboratory by artificial digestion and subjected to viability detection under a microscope. The extracted trichina muscle larvae were diluted with 0.9% normal saline and orally gavaged to a healthy sheep weighing 20 kg. After 60 days, the diaphragm muscle cut surface is planed and killed by a epidemic detection center, and is observed under a high light condition to find white spots with the size of a round oval needle point; the area with more white spots is cut by surgery, the size is about 20g, the area is put in a self-sealing bag after sterilization and disinfection, and microscopic examination and real-time fluorescence quantitative PCR are carried out in a laboratory. The focal part with the size of wheat grains is cut along the direction of muscle fiber by clamping with a bow-shaped scissors, the focal part is attached to a glass slide, a cover glass is covered, and two ends of the focal part are pinched to flatten tissues. When observed under a microscope, the shape was found to be olive-shaped trichina, as shown in fig. 9. The microscopic examination of the tissue is subjected to real-time fluorescence quantitative PCR, a sample group, a negative group and a blank control group are established, and the result is that the sample tissue is detected to be positive, as shown in figure 10.
6. Detecting suspected disease materials
Slaughterhouse sheep were numbered. According to the numbers, respectively taking left and right diaphragmatic tissues on each sheep body, removing diaphragmatic membranes, stretching or flattening the diaphragmatic tissues, and observing the oblique section of the diaphragmatic muscle under a high light condition to find out the point or abnormal meat-like tissue with suspected round oval color white on the diaphragmatic muscle. 73 sheep suspected tissues were collected in total, and the number of the suspected samples was 1 to 73. Each sample was taken in the laboratory with oval small white spots or abnormal mutton sheep tissue, each of approximately 2mm 10ram in size. The tissue mass is placed between two glass sheets and forced into a translucent shape. Four such sample tissues were taken for microscopy in each case. And 3 samples are checked to be negative by a mirror. DNA was extracted from the microscopic sheep diaphragmatic tissue and subjected to real-time fluorescent quantitative PCR. Wherein, the results of the numbers 8, 14, 32 and 37 are positive, the number 27 is weak positive, the results of the numbers 8 and 14 are shown in fig. 11, the results of the numbers 32, 37 and 27 are shown in fig. 12. The method is proved to be more sensitive.
The method of the invention designs a pair of specific primers by using the unknown Scfld4 gene of the chromosome of the trichina iss195 strain and establishes a method for detecting the trichina by using the DNA of the trichina as a template and real-time fluorescence quantitative PCR. The established standard curve is well fitted, and the standard curve is: y= -3.28x+23.531, correlation coefficient R 2 =0.9993, the amplification efficiency was 101.7%.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (3)
1. A real-time fluorescent quantitative PCR method for detecting trichina in sheep meat products is characterized in that,
the method comprises the following steps:
designing a primer according to the Scfld4 gene of the trichina iss195 strain chromosome;
diluting the extracted trichina genome DNA by a multiple ratio concentration, and respectively carrying out real-time fluorescence quantitative PCR detection on the trichina genome DNA sample diluted by the multiple ratio concentration by using the primers;
establishing a standard curve by taking CT values of the trichina genome DNA real-time fluorescence quantitative PCR detection diluted by the multiple ratio concentration as an ordinate and taking logarithms of corresponding DNA concentrations as an abscissa;
detecting a sample to be detected by using real-time fluorescence quantitative PCR, and calculating the corresponding concentration of the trichina genome DNA in the sample to be detected according to the CT value of the sample to be detected and the standard curve;
the sample is trichina genomic DNA;
the trichina is T1 type trichina;
the amplification primers of the Scfld4 gene are as follows:
an upstream primer: 5'-TTCTTAGTGCTTTCTGGGTCATCTT-3';
a downstream primer: 5'-CGCTTTTCCACTTACCATTCTGT-3';
the PCR reaction conditions are as follows: 95℃for 10min,95℃for 10s,60℃for 10s,72℃for 30s,40cycles;
the PCR reaction system is as follows: 1 XMonamp TM Chemoshs qPCR Mix 10 μl; 0.2. Mu.M upstream primer 0.4. Mu.L; 0.2. Mu.M downstream primer 0.4. Mu.L; 1 XLow ROX Dye or High ROX Dye 0.2. Mu.L; 9 mu L of DNA template; nuclear-Free Water to 20. Mu.L.
2. The method for detecting trichina in sheep meat products according to claim 1, wherein,
the diluted concentrations of the trichina genome DNA are respectively as follows: 41.10 ng/. Mu.L, 8.22 ng/. Mu.L, 1.644 ng/. Mu.L, 0.3288 ng/. Mu.L, 0.06576 ng/. Mu.L, 0.013152 ng/. Mu.L, 0.002630 ng/. Mu.L.
3. The method for detecting trichina in sheep meat products according to claim 1, wherein,
the standard curve is: y= -3.28x+23.531;
wherein Y is more than or equal to 18 and less than or equal to 35.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101381767A (en) * | 2008-10-24 | 2009-03-11 | 东北农业大学 | Universal real time fluorescent PCR detection method of trichinella |
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| CN101381767A (en) * | 2008-10-24 | 2009-03-11 | 东北农业大学 | Universal real time fluorescent PCR detection method of trichinella |
Non-Patent Citations (3)
| Title |
|---|
| Ananlysis of a 43-kDa glycoprotein from the intracellular parasitic nematode Trichinella spiralis;Pengfei Liu;《《Journal of Biological Chemistry》》;第第267卷卷(第第26期期);摘要，讨论，第18459-18465页 * |
| 张子群 ; 谢晓峰 ; 袁金钱 ; 由轩 ; 徐义刚 ; 单琳琳 ; 宋铭忻 ; .旋毛虫实时荧光PCR检测方法的建立与应用研究.检验检疫学刊.2009,(第03期),全文. * |
| 旋毛虫实时荧光PCR检测方法的建立与应用研究;张子群;谢晓峰;袁金钱;由轩;徐义刚;单琳琳;宋铭忻;;检验检疫学刊(第03期);全文 * |
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