CN111118189A - Bovine cryptosporidium parvum specific primer and PCR detection method and application thereof - Google Patents
Bovine cryptosporidium parvum specific primer and PCR detection method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of parasite detection, in particular to a bovine cryptosporidium parvum specific primer and a PCR detection method and application thereof. The method can quickly, sensitively and specifically detect the cryptosporidium parvum in the sample. The specific primer sequence of the bovine cryptosporidium parvum is as follows: upstream primer CpF: 5'-AGTGGTTACAGGTGGGATGAGT-3', respectively; the downstream primer CpR: 5'-GCGAGTTTCCTTGATTCATAGC-3', respectively; the common PCR and nano PCR detection method of the primer comprises the following steps: firstly, extracting DNA of the cryptosporidium parvum from a sample to be detected, then using the DNA as a template, carrying out PCR amplification by using a specific primer and carrying out electrophoresis detection on an amplification product, wherein if a specific band of about 410bp appears in an electrophoresis result, the existence of the bovine cryptosporidium parvum in the sample is indicated.
Description
The technical field is as follows:
the invention relates to the technical field of parasite detection, in particular to a bovine cryptosporidium parvum specific primer and a PCR detection method and application thereof.
Secondly, background art:
cryptosporidiosis in cattle is a highly harmful parasitic disease caused by the parasitism of Cryptosporidium (Cryptosporidium) in the epithelial cells of the bovine gastrointestinal tract. The research shows that the common cryptosporidium of cattle has 4 kinds, namely cryptosporidium parvum (C.parvum), cryptosporidium andersoni (C.andersoni), bovine cryptosporidium bovium (C.bovis) and cryptosporidium raperii (C.ryana). Among them, cryptosporidium parvum is an important zoonosis species, and seriously threatens the health of human and livestock. In China, the infection rate of C.parvum of milk cows before weaning in certain areas can reach 31.4 percent, and severe watery diarrhea of calves can be caused by the infection of the milk cows before weaning, so that the calves are seriously dehydrated and even die. Adult cattle infected with the feed also lose weight for a short time, and the production performance is impaired, thereby causing serious economic loss to the animal husbandry production. More importantly, cattle, especially calves, play a major role in the intercourse of cryptosporidiosis in animals and humans, and cattle infected with c.parvum can spread pathogens through feces, contaminate feed, drinking water, and contaminate milk sources, becoming one of the important causes of human outbreaks of food-and water-borne cryptosporidiosis.
Currently, the clinical diagnosis of cryptosporidiosis in cattle mainly comprises a saturated sucrose flotation method based on oocysts in feces, an improved acid-fast staining method, a fluorescent antibody staining method and a nested PCR method. Although the saturated sucrose floating method and the improved acid-fast staining method based on morphology and microscopic observation are simple and convenient, the sensitivity and specificity are poor, professional personnel are required, and the method is not suitable for basic clinical application; fluorescent antibody staining, while relatively sensitive and specific, requires expensive fluorescent microscopes and antibodies. Nested PCR technology is widely used for the detection of bovine Cryptosporidium with its high specificity and sensitivity. However, this method is cumbersome, requires two rounds of PCR amplification, is time and labor intensive, and requires sequencing techniques to identify the species.
In recent years, the common PCR method is widely applied to the aspect of laboratory pathogen detection because the common PCR method is suitable for detecting most pathogens, the detection result is more accurate, and the prices of corresponding equipment and reagents are lower than those of other PCR methods. In addition, with the increasing development of nanotechnology and the wide application in molecular biology, nano-PCR has come into play. The principle of nano PCR is that nano particles are added into a common PCR system, and the excellent thermal conductivity of the nano particles is utilized to shorten the heating and cooling time in the amplification process, so that the whole reaction system reaches the equilibrium temperature in the shortest time, thereby improving the amplification efficiency; more importantly, the nanoparticles can be combined with single-stranded DNA to play a role similar to single-stranded binding protein SSB, so that the mismatching of a primer and a template is obviously reduced, the specificity of PCR reaction is improved, and the nonspecific amplification is effectively reduced. In 2005, the first report that nanogold could significantly improve PCR non-specific amplification and was effective over a wider temperature range was made by the institute of physical application and research in the department of chinese academy of sciences and the university of shanghai transportation; a large number of test results of other researchers show that the sensitivity of the nano PCR is 100-1000 times higher than that of the common PCR.
Third, the invention
The invention provides a specific primer of bovine cryptosporidium parvum, a PCR detection method and application thereof, which respectively establish a common PCR and nano PCR detection method of bovine cryptosporidium parvum by utilizing a self-designed specific primer and optimizing reaction conditions, and aims to provide a faster, more accurate and more efficient pathogen detection method for clinical diagnosis of bovine cryptosporidium parvum.
In order to achieve the purpose, the invention adopts the technical scheme that: a bovine cryptosporidium parvum specific primer is characterized in that: the specific primer sequences are as follows:
upstream primer CpF: 5'-AGTGGTTACAGGTGGGATGAGT-3', respectively;
the downstream primer CpR: 5'-GCGAGTTTCCTTGATTCATAGC-3' are provided.
The application of the specific primer in detecting the bovine cryptosporidium parvum.
The common PCR detection method of the specific primer in the detection of the bovine cryptosporidium parvum comprises the following steps:
1) extracting DNA of a sample to be detected to obtain an amplification template;
2) carrying out PCR amplification on the DNA sample obtained in the step 1) by using a specific primer, wherein a PCR reaction system is as follows: 10 Xex Taq buffer (Mg)2+free)1.25μL，MgCl20.6. mu.L (25mM), 1. mu.L of dNTP (2.5mM), 0.5. mu.L of each of upstream and downstream primers (10mM), 0.07. mu.L of ex Taq enzyme (5U/. mu.L), 1. mu.L of template DNA, ddH2Supplementing O to 12 mu L; the reaction conditions are as follows: 5min at 94 ℃; 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃;
3) and (3) electrophoretic detection of PCR products: and detecting the amplification product by 1% agarose gel electrophoresis, wherein if a specific band of about 410bp appears in the electrophoresis result, the cryptosporidium parvum exists in the sample to be detected.
The nano PCR detection method for detecting the bovine cryptosporidium parvum by using the specific primers comprises the following steps:
1) extracting DNA of a sample to be detected to obtain an amplification template;
2) carrying out nano PCR amplification on the DNA sample obtained in the step 1) by using a specific primer, wherein the reaction system is as follows: 2 XNano-QPCR buffer 6. mu.L, upstream and downstream primers (10mM) 1. mu.L each, Taq enzyme mix (5U/. mu.L) 0.2. mu.L, template DNA 1. mu.L, ddH2Supplementing O to 12 mu L; the reaction conditions are as follows: 5min at 94 ℃; 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃;
3) and (3) electrophoretic detection of PCR products: and detecting the amplification product by 1% agarose gel electrophoresis, wherein if a specific band of about 410bp appears in the electrophoresis result, the cryptosporidium parvum exists in the sample to be detected.
The PCR detection method is applied to the identification, diagnosis and prevention of bovine cryptosporidiosis.
Compared with the prior art, the invention has the following advantages and effects:
1) the invention establishes the common PCR detection method of the bovine cryptosporidium parvum, the operation of the method requires relatively low prices of instruments and reagents, the common molecular biology laboratory can complete clinical detection, the operation is simple and convenient, the result is more accurate, and the method has good universal applicability and operability in clinical application;
2) the invention establishes the nano PCR detection method of the bovine cryptosporidium parvum, and the nano particles are added into the reaction system, so that the target temperature can be reached more quickly, the time for the whole system to reach the temperature balance is reduced, and the nano PCR detection method has better sensitivity and specificity;
3) the common PCR and nano PCR method established by the invention provides a reliable technical means for the clinical detection of the bovine cryptosporidium parvum, provides technical support for the screening, purification and comprehensive prevention of the bovine cryptosporidium parvum, and can quickly, sensitively and specifically detect the cryptosporidium parvum in a sample;
4) clinical tests show that the specific primer provided by the invention can detect the bovine cryptosporidium parvum, and has a negative result on other cryptosporidium parvum infected by cattle, and the primer specificity is better.
Fourthly, explanation of the attached drawings:
FIG. 1 shows the result of conventional PCR amplification electrophoresis; m: DL2000DNA Marker; 1: cryptosporidium parvum DNA; 2: negative control;
FIG. 2 shows the result of optimizing the annealing temperature of a conventional PCR; m: DL2000DNA Marker; 1-8: 50 deg.C, 50.5 deg.C, 52 deg.C, 54 deg.C, 56 deg.C, 58 deg.C, 59.5 deg.C, 60 deg.C; 9: negative control;
FIG. 3 shows the result of optimizing the amount of magnesium ions in PCR; m: DL2000DNA Marker; 1-7: 0.2. mu.L, 0.4. mu.L, 0.6. mu.L, 0.8. mu.L, 1.0. mu.L, 1.2. mu.L, 1.4. mu.L; 8: negative control;
FIG. 4 shows the optimization results of the annealing temperature of nano PCR; m: DL2000DNA Marker; 1-8: 50 deg.C, 50.5 deg.C, 52 deg.C, 54 deg.C, 56 deg.C, 58 deg.C, 59.5 deg.C, 60 deg.C; 9: negative control;
FIG. 5 shows the optimized result of the usage of the nano PCR primers; m: DL2000DNA Marker; 1-7: 2pmol, 4pmol, 6pmol, 8pmol, 10pmol, 12pmol, 14 pmol; 8: negative control;
FIG. 6 shows the results of sensitivity tests of ordinary PCR and nano-PCR; a: electrophoresis picture of general PCR amplification product; b: electrophoresis picture of nano PCR amplification product. M: DL2000DNA Marker; 1: 102 ng; 2: 10.2 ng; 3: 1.02 ng; 4: 102 pg; 5: 10.2 pg; 6: 1.02 pg; 7: 102 fg; 8: 10.2 fg; 9: 1.02 fg; 10: negative control;
FIG. 7 shows the specificity test results of general PCR and nano-PCR; a: electrophoresis picture of general PCR amplification product; b: electrophoresis picture of nano PCR amplification product. M: DL2000DNA Marker; 1: cryptosporidium parvum; 2: cryptosporidium andersoni; 3: cryptosporidium bovis; 4: cryptosporidium raperii; 5: blastocyst protozoa; 6: giardia lamblia; 7: intestinal microsporidia peelii; 8: ciliates of the colon; 9: negative control;
FIG. 8 shows the results of the nested PCR (GP60), the ordinary PCR and the nano PCR on clinical samples; a: electrophoresis picture of nest PCR (GP60) amplification product; b: electrophoresis picture of general PCR amplification product; c: electrophoresis picture of nano PCR amplification product. M: DL2000DNA Marker; 1-20: calf feces samples; 21: and (5) negative control.
The fifth embodiment is as follows:
a specific primer sequence of the bovine cryptosporidium parvum is as follows:
upstream primer CpF: 5'-AGTGGTTACAGGTGGGATGAGT-3', respectively;
the downstream primer CpR: 5'-GCGAGTTTCCTTGATTCATAGC-3' are provided.
The specific primer is applied to detecting the bovine cryptosporidium parvum.
The common PCR detection method of the specific primer in detecting the bovine cryptosporidium parvum comprises the following steps:
1) extracting DNA of a sample to be detected to obtain an amplification template;
2) carrying out PCR amplification on the DNA sample obtained in the step 1) by using a specific primer, wherein a PCR reaction system is as follows: 10 Xex Taq buffer (Mg)2+free)1.25μL，MgCl20.6. mu.L (25mM), 1. mu.L of dNTP (2.5mM), 0.5. mu.L of each of upstream and downstream primers (10mM), 0.07. mu.L of ex Taq enzyme (5U/. mu.L), 1. mu.L of template DNA, ddH2Supplementing O to 12 mu L; the reaction conditions are as follows: 94 ℃ for 5min(ii) a 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃;
3) and (3) electrophoretic detection of PCR products: and detecting the amplification product by 1% agarose gel electrophoresis, wherein if a specific band of about 410bp appears in the electrophoresis result, the cryptosporidium parvum exists in the sample to be detected.
The nano PCR detection method for detecting the bovine cryptosporidium parvum by using the specific primers comprises the following steps of:
1) extracting DNA of a sample to be detected to obtain an amplification template;
2) carrying out nano PCR amplification on the DNA sample obtained in the step 1) by using a specific primer, wherein the reaction system is as follows: 2 XNano-QPCR buffer 6. mu.L, upstream and downstream primers (10mM) 1. mu.L each, Taq enzyme mix (5U/. mu.L) 0.2. mu.L, template DNA 1. mu.L, ddH2Supplementing O to 12 mu L; the reaction conditions are as follows: 5min at 94 ℃; 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃;
3) and (3) electrophoretic detection of PCR products: and detecting the amplification product by 1% agarose gel electrophoresis, wherein if a specific band of about 410bp appears in the electrophoresis result, the cryptosporidium parvum exists in the sample to be detected.
The PCR detection method is applied to the identification, diagnosis and prevention of bovine cryptosporidiosis.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.
Example 1:
establishment of common PCR and nano PCR detection method for bovine cryptosporidium parvum
Firstly, primer design and synthesis:
primers were designed with reference to the locus of the Cryptosporidium parvum Iowa II cultly Gene (Gene ID:3373948) published in GenBank, and the reference sequences were as follows:
using the software DNAMAN 7, specific primers for synthesizing the Cryptosporidium parvum Iowa II cultly Gene (Gene ID:3373948) were selected based on the sequence of the Cryptosporidium parvum Iowa II cultly Gene, the primers were synthesized by Biotechnology engineering (Shanghai) GmbH, and the sequences of the primers are shown in Table 1.
TABLE 1 Cryptosporidium parvum PCR amplification specific primers
Second, establishment of ordinary PCR method
(1) The general PCR reaction system is preset to be 10 Xex Taq buffer (Mg)2+free)1.25μL，MgCl2mu.L (25mM), 1. mu.L dNTP (2.5mM), 0.5. mu.L each of upstream and downstream primers (10mM), 0.07. mu.L of ex Taq enzyme (5U/. mu.L), 1. mu.L of template DNA, ddH2O is supplemented to 12 mu L. The reaction conditions are preset to 94 ℃ for 5 min; 45s at 94 ℃, 30s at 58 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃. And amplifying the DNA of the cryptosporidium parvum by using a preset PCR. The amplified product was detected by 1% agarose gel electrophoresis, and as a result, the desired amplified fragment appeared at about 410bp, as shown in FIG. 1. The amplified product was sent to Biotechnology engineering (Shanghai) GmbH for sequencing, and sequence alignment analysis showed that the similarity to the Cryptosporidium parvum Iowa II cultly gene (GeneID:3373948) was 99.47%, which is the target amplified fragment.
The sequence of the amplification product:
(2) optimization of ordinary PCR annealing temperature
PCR amplification was carried out at annealing temperatures of 50, 50.5, 52, 54, 56, 58, 59.5 and 60 ℃ by setting a temperature gradient according to the TM values of the upstream and downstream primers, and the other conditions were the same as those set in (1). The electrophoresis results are shown in FIG. 2, and the amplified band is brightest when the annealing temperature is 56 ℃.
(3) Optimization of magnesium ion concentration in general PCR
0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mu L MgCl are respectively added into the system2(25mM) and PCR amplification was performed at the optimized annealing temperature under the same conditions as set forth in (1). As shown in FIG. 3, the band obtained by electrophoresis was brightest when the amount of magnesium ions was 0.6. mu.L.
Through the optimization of annealing temperature and magnesium ion concentration, the general PCR method is finally determined as a reaction system: 10 Xex Taqbuffer (Mg)2+free)1.25μL，MgCl20.6. mu.L (25mM), 1. mu.L of dNTP (2.5mM), 0.5. mu.L of each of upstream and downstream primers (10mM), 0.07. mu.L of ex Taq enzyme (5U/. mu.L), 1. mu.L of template DNA, ddH2O is supplemented to 12 mu L. Reaction conditions are as follows: 5min at 94 ℃; 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃.
Establishment of three, nanometer PCR method
(1) Optimization of nano PCR annealing temperature
The nano PCR reaction system is preset as follows: 2 XNano-QPCR buffer 6. mu.L, upstream and downstream primers (10mM) 1. mu.L each, Taq enzyme mix (5U/. mu.L) 0.2. mu.L, template DNA 1. mu.L, ddH2O to 12. mu.L. The reaction conditions were preset as follows: the reaction condition is 94 ℃ for 5 min; 45s at 94 ℃, 30s at 58 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃.
Temperature gradients were set according to the TM values of the upstream and downstream primers, and PCR amplification was performed at annealing temperatures of 50, 50.5, 52, 54, 56, 58, 59.5, and 60 ℃ respectively, under the same conditions as set in advance. The electrophoresis results are shown in FIG. 4, and the amplified band is brightest when the annealing temperature is 56 ℃.
(2) Optimization of nano PCR primer dosage
2, 4, 6, 8, 10, 12 and 14pmol of primers are respectively added into the system, nano PCR amplification is carried out at the optimized annealing temperature, and other conditions are preset to be the same as those in the step (1). As a result of electrophoresis, as shown in FIG. 5, when the amount of the primer was 10pmol (i.e., 1. mu.L of the primer at a concentration of 10mM), the amplified band was brightest.
By optimizing the annealing temperature and the primer amount, the nano PCR method is finally determined as a reaction system: 2 XNano-QPCRbuffer 6. mu.L, upstream and downstream primers (10mM) 1. mu.L each, Taq enzyme mix (5U/. mu.L) 0.2. mu.L, template DNA 1. mu.L, ddH2O to 12. mu.L. Reaction conditions are as follows: 5min at 94 ℃; 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃.
Sensitivity test of common PCR and nano PCR detection method
And (3) measuring the mass concentration of the extracted DNA of the cryptosporidium parvum to be 102 ng/mu L by using a micro-spectrophotometer, diluting the DNA to be 1.02 fg/mu L by using a 10-fold ratio, amplifying the template concentration gradient by respectively utilizing an established nano PCR method and a common PCR method, and comparing the sensitivity of the two methods.
The electrophoresis result is shown in FIG. 6, where the minimum template detection amount of ordinary PCR is 102pg, the minimum template detection amount of nano-PCR is 102fg, and the sensitivity of nano-PCR is 1000 times that of ordinary PCR.
Fifth, specificity test of common PCR and nano PCR detection method
And amplifying DNA of cryptosporidium parvum, cryptosporidium andersoni, cryptosporidium bovis, cryptosporidium rapae, blastocystis, Giardia lamblia, Microsporidium piperita and Giardia coli by using the optimized common PCR and nano PCR, and verifying the specificity of the established detection method. The electrophoresis result is shown in figure 7, after the DNA sample is amplified by nano PCR, a specific band of about 410bp appears only in the cryptosporidium parvum, and a non-specific amplification band of about 800bp appears in the Pediosporidium bicucum by common PCR.
Comparing the detection results of the nested PCR detection method, the common PCR detection method and the nano PCR detection method on clinical samples:
and respectively carrying out cryptosporidium parvum nested PCR (GP60), common PCR and nano PCR detection on 20 clinically collected calf feces samples, and comparing detection results.
As shown in FIG. 8, the positive rate of Cryptosporidium parvum in the test sample was 25% (5/20) by nested PCR amplification; after the ordinary PCR and the nano PCR amplification, the positive rate of the cryptosporidium parvum of the sample is 30 percent (6/20). The PCR products with negative nested PCR detection and positive ordinary PCR and nano-PCR detection are sent to the company Limited in Biotechnology engineering (Shanghai) for sequencing, and the similarity with the Cryptosporidium parvum Iowa II cultly Gene (Gene ID:3373948) is 98.71 percent through sequence comparison, which indicates that the positive result is not false positive.
Amplification product sequence
Compared with the nested PCR detection method, the common PCR detection method and the nano PCR detection method established by the invention can obtain a detection result only by one-time amplification, save time and reagent consumption, have higher detection rate and are suitable for screening the bovine cryptosporidium parvum of clinical samples. Compared with the common PCR, the nano PCR can reach the target temperature more quickly during amplification, the amplification efficiency is improved, the non-specific amplification is effectively reduced, and the interference of false positive results on diagnosis is reduced.
In conclusion, the common PCR and nano PCR method established by the invention has the advantages of rapidness, strong sensitivity and high specificity on the detection of the bovine cryptosporidium parvum, can provide an effective detection method for the clinical diagnosis of the bovine cryptosporidium parvum, and provides technical support for the screening, purification and comprehensive control of bovine cryptosporidiosis in cattle.
The above description is only for the preferred embodiment of the present invention, and not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made to the technical solutions or parts of the technical features described in the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> northwest agriculture and forestry science and technology university
<120> bovine cryptosporidium parvum specific primer and PCR detection method and application thereof
<130>2020
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Claims (5)
1. A bovine cryptosporidium parvum specific primer is characterized in that: the specific primer sequences are as follows:
upstream primer CpF: 5'-AGTGGTTACAGGTGGGATGAGT-3', respectively;
the downstream primer CpR: 5'-GCGAGTTTCCTTGATTCATAGC-3' are provided.
2. The use of the specific primer as claimed in claim 1 for detecting bovine cryptosporidium parvum.
3. The general PCR detection method of the specific primers in detecting bovine cryptosporidium parvum according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
1) extracting DNA of a sample to be detected to obtain an amplification template;
2) carrying out PCR amplification on the DNA sample obtained in the step 1) by using a specific primer, wherein a PCR reaction system is as follows: 10 XexeTaq buffer (Mg)2+free)1.25μL，MgCl20.6. mu.L (25mM), 1. mu.L of dNTP (2.5mM), 0.5. mu.L of each of upstream and downstream primers (10mM), 0.07. mu.L of ex Taq enzyme (5U/. mu.L), 1. mu.L of template DNA, ddH2Supplementing O to 12 mu L; the reaction conditions are as follows: 5min at 94 ℃; 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃;
3) and (3) electrophoretic detection of PCR products: and detecting the amplification product by 1% agarose gel electrophoresis, wherein if a specific band of about 410bp appears in the electrophoresis result, the cryptosporidium parvum exists in the sample to be detected.
4. The nano PCR detection method for detecting bovine cryptosporidium parvum by using the specific primers as claimed in claim 1, wherein the specific primers comprise the following components: the method comprises the following steps:
1) extracting DNA of a sample to be detected to obtain an amplification template;
2) carrying out nano PCR amplification on the DNA sample obtained in the step 1) by using a specific primer, wherein the reaction system is as follows: 2 XNano-QPCR buffer 6. mu.L, upstream and downstream primers (10mM) 1. mu.L each, Taq enzyme mix (5U/. mu.L) 0.2. mu.L, template DNA 1. mu.L, ddH2Supplementing O to 12 mu L; the reaction conditions are as follows: 5min at 94 ℃; 45s at 94 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃;
3) and (3) electrophoretic detection of PCR products: and detecting the amplification product by 1% agarose gel electrophoresis, wherein if a specific band of about 410bp appears in the electrophoresis result, the cryptosporidium parvum exists in the sample to be detected.
5. Use of the PCR detection method of claim 3 or 4 for the identification, diagnosis and prevention of bovine cryptosporidiosis.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6153411A (en) * | 1998-10-30 | 2000-11-28 | American Water Works Company, Inc. | Methods and kits for detection of Cryptosporidium parvum using immunomagnetic separation and amplification |
| JP2003033184A (en) * | 2001-07-18 | 2003-02-04 | Goda Hiroshi | Kit and method for cryptosporidium detection |
| WO2006044045A2 (en) * | 2004-09-07 | 2006-04-27 | Virginia Commonwealth University | Cryptosporidium hominis genes and gene products for chemotherapeutic, immunoprophylactic and diagnostic applications |
| CN1814806A (en) * | 2005-12-08 | 2006-08-09 | 华南农业大学 | C. andersoni PCR detecting kit |
| AU2007203610A1 (en) * | 2000-09-12 | 2007-08-23 | Gen-Probe Incorporated | Compositions, methods and kits for determining the presence of cryptosporidium organisms in a test sample |
| CN101353690A (en) * | 2008-07-11 | 2009-01-28 | 吉林大学 | Cryptosporidium and cryptosporidium parvum specific PCR detecting reagent kit and detecting method |
| CN103898203A (en) * | 2012-12-28 | 2014-07-02 | 中国科学院动物研究所 | Method for detecting cryptosporidium parvum and detection kit |
| CN107058619A (en) * | 2017-03-10 | 2017-08-18 | 中国农业科学院特产研究所 | Bovine Respiratory Syncytial virus nano PCR detection kits and preparation method thereof |
| CN108796106A (en) * | 2018-06-29 | 2018-11-13 | 吉林大学 | Giardia bovis, Cryptosporidium multiple PCR detection kit and its method |
| CN108893526A (en) * | 2018-03-07 | 2018-11-27 | 西北农林科技大学 | A kind of Cryptosporidum parvum fluorescence LAMP detection method |
| CN109234420A (en) * | 2018-10-09 | 2019-01-18 | 河南省奶牛生产性能测定中心 | Detect fluorescence quantification PCR primer, probe, kit and the method for Cryptosporidum parvum |
-
2020
- 2020-01-07 CN CN202010014664.9A patent/CN111118189A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6153411A (en) * | 1998-10-30 | 2000-11-28 | American Water Works Company, Inc. | Methods and kits for detection of Cryptosporidium parvum using immunomagnetic separation and amplification |
| AU2007203610A1 (en) * | 2000-09-12 | 2007-08-23 | Gen-Probe Incorporated | Compositions, methods and kits for determining the presence of cryptosporidium organisms in a test sample |
| JP2003033184A (en) * | 2001-07-18 | 2003-02-04 | Goda Hiroshi | Kit and method for cryptosporidium detection |
| WO2006044045A2 (en) * | 2004-09-07 | 2006-04-27 | Virginia Commonwealth University | Cryptosporidium hominis genes and gene products for chemotherapeutic, immunoprophylactic and diagnostic applications |
| CN1814806A (en) * | 2005-12-08 | 2006-08-09 | 华南农业大学 | C. andersoni PCR detecting kit |
| CN101353690A (en) * | 2008-07-11 | 2009-01-28 | 吉林大学 | Cryptosporidium and cryptosporidium parvum specific PCR detecting reagent kit and detecting method |
| CN103898203A (en) * | 2012-12-28 | 2014-07-02 | 中国科学院动物研究所 | Method for detecting cryptosporidium parvum and detection kit |
| CN107058619A (en) * | 2017-03-10 | 2017-08-18 | 中国农业科学院特产研究所 | Bovine Respiratory Syncytial virus nano PCR detection kits and preparation method thereof |
| CN108893526A (en) * | 2018-03-07 | 2018-11-27 | 西北农林科技大学 | A kind of Cryptosporidum parvum fluorescence LAMP detection method |
| CN108796106A (en) * | 2018-06-29 | 2018-11-13 | 吉林大学 | Giardia bovis, Cryptosporidium multiple PCR detection kit and its method |
| CN109234420A (en) * | 2018-10-09 | 2019-01-18 | 河南省奶牛生产性能测定中心 | Detect fluorescence quantification PCR primer, probe, kit and the method for Cryptosporidum parvum |
Non-Patent Citations (4)
| Title |
|---|
| ABRAHAMSEN,M.S. 等: "Cryptosporidium parvum Iowa II chromosome 3, whole genome shotgun sequence", 《GENEBANK》 * |
| C MARY 等，: "Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium", 《MULTICENTER STUDY J CLIN MICROBIOL》 * |
| 丁慧萍等: "微小隐孢子虫的基因检测实验研究", 《中国寄生虫病防治杂志》 * |
| 朱正 等: "犬巴贝斯虫和犬埃立克体双重纳米PCR方法的建立及初步应用", 《2019中国畜牧兽医学会兽医外科学分会第十届会员***暨第24次学术研讨会论文集中国畜牧兽医学会会议论文集》 * |
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