CN116445641A - PCR detection method for Pityrosporum tobermori - Google Patents
PCR detection method for Pityrosporum tobermori Download PDFInfo
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Abstract
The invention discloses a PCR detection method for pirimiphos, which relates to the technical field of PCR detection and has the technical key points that: the method comprises the following steps: s1, sample collection: collecting 24 parts of blood of 24 cattle and sheep, respectively placing in anticoagulant tubes, and storing at-20deg.C; s2, designing a primer and extracting whole blood DNA: s3, amplification of PCR: s4, constructing positive plasmids: s5, extracting DNA from the collected cattle and sheep blood clinical samples according to the steps S1-S4, and detecting piroplasmosis and performing agarose gel electrophoresis. The method adopts 18SrRNA design 1 of piriform worms to carry out optimization of reaction conditions by taking positive nucleic acid as a template and detection of sensitivity, specificity and clinical samples; the method is sensitive, quick, simple and convenient, has high specificity, and can be used for quick detection and diagnosis of piroplasmosis and epidemiological investigation.
Description
Technical Field
The invention relates to the technical field of PCR detection, in particular to a method for detecting piroplasmosis PCR.
Background
Piroplasmosis (piroplasmosis) including Theileria (Theileria) and Babesia (Babesia), are blood protozoa parasitic in animal erythrocytes and can cause theileriosis and babesiasis, which are distributed globally, and are frequent in tropical and subtropical areas. Clinically, the Chinese medicinal composition has symptoms of high fever, anemia, jaundice, bleeding, dyspnea and the like, and has extremely high death rate. Piriosis is a transmission medium for ticks, so the epidemic of the disease has certain seasonality and locality.
The diagnosis of piroplasmosis mainly dependent on clinical examination and blood smear detection, and although the piroplasmosis is simple and easy to operate, the comparison depends on the experience of detection personnel, and the detection accuracy cannot be ensured. In addition, serological methods can be used for detection of Pirion, but are limited by antigen sources and cross-reactivity. With the advent of molecular biology methods, rapid, accurate and highly sensitive PCR methods are becoming the primary detection method. At present, the common detection methods for piroplasmosis in China are mostly nest type PCR, the time is long, the method is not suitable for detecting samples in a large scale, and most common PCR detection methods can not detect theileria and babesia simultaneously.
Therefore, it is important to establish an accurate and rapid pirimiphos PCR detection method, and the present invention aims to provide a pirimiphos PCR detection method to solve the above problems.
Disclosure of Invention
The invention aims to solve the problems and provide a PCR detection method for piroplasmosis, the method designs 1 pair of specific primers according to theileria taylori 18S rRNA, and establishes a PCR detection method with strong specificity and high sensitivity by optimizing the conditions of the PCR method, thereby providing a rapid, simple and efficient detection method for monitoring piroplasmosis.
In order to achieve the above purpose, the technical scheme of the invention is as follows: a PCR detection method for Pityrosporum cucumerinum comprises the following steps:
s1, sample collection: collecting 24 parts of blood of 24 cattle and sheep, respectively placing in anticoagulant tubes, and storing at-20deg.C;
s2, designing a primer and extracting whole blood DNA:
A. comparing the gene sequences of theileria inylanica 18S rRNA retrieved from NCBI by DNAStar7.1 software, and then designing universal primers P-1 and P-2 by adopting Premier6.0 software;
B. extracting the whole blood DNA of 24 parts of cattle and sheep blood in the step S1 by using a genome DNA purification kit, and storing the whole blood DNA at the temperature of-20 ℃ for later use;
s3, amplification of PCR:
1) PCR amplification is carried out by taking the DNA of the Theileria luwenshuni whole blood sample as a template and adopting a primer P-1/P-2, wherein the amplification system is as follows: 10 mu L of PCR premix, 6 mu L of dd H2O, 1 mu L of each of the upstream and downstream primers and 2 mu L of template DNA; the amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 61℃for 30s, extension at 72℃for 45s, and cycling for 35 times; extending at 72 ℃ for 8min;
2) Amplifying the Chinese theileria, the ring theileria, the Theileria ewingii, the babesia bifidus, the babesia moellendorffii, the babesia ovale and the babesia bovis respectively by using a primer P-1/P-2, detecting PCR products by using 1.2% agarose gel electrophoresis, and verifying whether the primer can amplify the Piroparrangement;
s4, constructing positive plasmids:
placing the agarose gel after electrophoresis in the step S3 in an ultraviolet analyzer, cutting off a target strip, placing the cut target strip in an EP tube with the volume of 1.5mL, then performing the operation of a DNA gel recovery kit, and finally measuring the concentration of a gel recovery product; the gel recovery product is connected with a pMD18-T carrier and then is transformed into DH-5 alpha competent cells, single bacterial colonies are selected through screening of ampicillin positive LB culture medium, and are subjected to shake cultivation for 3-5 hours at 37 ℃ and 200r/min, the obtained turbid bacterial liquid is used for extracting plasmids from bacterial liquid by using a kit, the concentration of the plasmids is measured by using a spectrophotometer, sequencing is carried out, whether the plasmids are target products or not is identified, and the residual plasmids are mixed with glycerol and then stored for standby; the plasmid identified as the product of interest was designated pMD-TH;
s5, extracting DNA from the collected cattle and sheep blood clinical samples according to the steps S1-S4, and detecting piroplasmosis and performing agarose gel electrophoresis.
Further, the primer sequence in step S2 is:
P-1：5＇-CGGCTACCACATCTAAGGAAGG-3＇；
P-2：5＇-CAAGGTGCTGAAGGAGTCGTAA-3＇。
further, the gene sequences for alignment of the taylor 18S rRNA gene sequences retrieved from NCBI in step S2 include taylor MH208642.1, taylor MF287948.1, taylor ewing JF719835.1, taylor sheep MN493111.1, taylor LC326007.2, bifidus ba Bei Sichong MT539381.1, dual bud ba Bei Sichong KY805824.1, babesia bovis KP710223.1, babesia ovale MN900525.1.
Compared with the prior art, the beneficial effect of this scheme:
1. the method adopts 18S rRNA design 1 of piriform worms to carry out optimization of reaction conditions by taking positive nucleic acid as a template and detection of sensitivity, specificity and clinical samples;
2. the method is sensitive, quick, simple and convenient, has high specificity, and can be used for quick detection and diagnosis of piroplasmosis and epidemiological investigation.
Drawings
FIG. 1 shows the results of PCR amplification in examples of the present invention (1 is a positive sample; 2 is a negative control);
FIG. 2 is a verification of the primer range for Piroparrangement in the examples of the present invention (1, theileria sinensis; 2, theileria annulata; 3, theileria euglena; 4, theileria luwensis; 5, theileria orientalis; 6, bulbeida bisporus; 7, bulbeida moensis; 8, bulbeida ovalis; 9, bulbeida bovis);
FIG. 3 shows the optimization of the annealing temperature in the examples of the present invention (1.51 ℃;2.53 ℃;3.55 ℃;4.57 ℃;5.59 ℃;6.61 ℃;7.63 ℃;8.65 ℃;9.67 ℃;10.69 ℃;11.71 ℃;12.73 ℃;13.75 ℃).
FIG. 4 is a specificity test (1. Positive sample; 2. Trypanosoma evanescens; 3. No slurry; 4. Hepatic clusterin; 5. Toxoplasma; 6. Erlike) in an example of the invention;
FIG. 5 is a sensitivity test (10-fold dilution of 1-11 in order) for Piroprion in the examples of the present invention;
FIG. 6 shows the clinical sample detection of PCR in the examples of the present invention (24 clinical samples 1-24);
FIG. 7 is a test of a control group of Piroprion clinical samples (24 clinical samples 1-24) in an example of the invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the illustrated embodiments are merely exemplary of some, but not all, of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
Examples:
the following are specific examples of the inventive protocol:
1. material
1.1, sample Source
Collecting 24 cattle and sheep blood in anticoagulation tube in Guangxi river basin area, and preserving at-20deg.C. The Theileria luwensis, theileria orientalis, trypanosoma evans, naemorhedi and Babesia pubescens positive DNA samples were all kept by the university of Guangxi animal science and technology institute parasite laboratory, the DNA samples of Theileria luwensis, theileria annulata, theileria uillarum, bulletia bifida, babesia morgans, babesia ovalis and Babesia bovis were given away by the laboratory of the animal institute of Lanzhou.
1.2 major reagents
Dream Taq Green PCR premix (2×), generule 100bp Plus DNA Ladder, genomic DNA purification kit, available from Thermo Fisher Scientific company; DL2000 Plus DNA Marker, available from Nanning Jieshi Biol Co., ltd; plasmid extraction kit and DNA gel recovery kit were purchased from Tiangen Biochemical technology (Beijing) Co., ltd; pMD18-T vector, available from Takara Bio Inc.
2. Method of
2.1 design of primers and extraction of Whole blood DNA
The Theileria 18S rRNA gene sequences retrieved from NCBI (Theileria eastern MH208642.1, theileria annulata MF287948.1, theileria ewingii JF719835.1, theileria ovis MN 493111.1), theileria reuteri LC326007.2, theileria bifidus Bei Sichong MT539381.1, double bud Bei Sichong KY805824.1, bulbeis bovis KP710223.1, oval bar Bei Sichong MN 900525.1) were aligned using DNAStar7.1 software, and the universal primers P-1 and P-2 were designed using Premier6.0 software (see Table 1). Extracting 24 parts of cattle and sheep whole blood DNA by using a genome DNA purification kit, and preserving at-20 ℃ for later use.
TABLE 1 design of primers
Table 1Primer design
2.2 amplification by PCR
The DNA of the whole blood sample of the Theileria luwenshuni preserved in the laboratory is used as a template, and the primer P-1/P-2 is used for PCR amplification, and the amplification system is as follows: 10 mu L of PCR premix and dd H 2 O6. Mu.L, 1. Mu.L of each of the upstream and downstream primers, and 2. Mu.L of the template DNA. Amplification procedure: pre-denatured at 94℃for 5min; denaturation at 94℃for 30s, annealing at 61℃for 30s, extension at 72℃for 45s, and cycling for 35 times; extending at 72℃for 8min.
P-1/P-2 was used to amplify Theileria sinensis, theileria annulata, theileria uligera, baseba bibud, baseba morse, baseba ovalis and Bulbeis bovis, and the PCR products were detected by 1.2% agarose gel electrophoresis, and it was verified whether the primers could amplify the Piroparrangement.
2.3 construction of Positive plasmids
The agarose gel after electrophoresis is placed in an ultraviolet analyzer, the target band is cut off and then placed in a 1.5mL EP tube, then the operation is carried out according to the instruction of the DNA gel recovery kit, and finally the concentration of the gel recovery product is measured. The gel recovery product is connected with a pMD18-T carrier and then is transformed into DH-5 alpha competent cells, a single colony is selected through screening of an ampicillin positive LB culture medium, shaking culture is carried out for 3-5 hours at 37 ℃ and 200r/min, the obtained turbid bacterial liquid is used for extracting plasmids from the bacterial liquid by using a kit, the concentration of the plasmids is measured by using a spectrophotometer, the plasmids are sent to Guangzhou Hua big gene science and technology Co-Ltd for sequencing, whether the plasmids are target products or not is identified, and the residual plasmids are mixed with glycerol and then stored for standby. The plasmid identified as correct was designated pMD-TH.
2.4 optimization of annealing temperature
Under other conditions, the PCR amplification was performed with P-1/P-2 at different temperatures of 51℃53℃55℃57℃59℃61℃63℃65℃67℃69℃71℃73℃75℃and analyzed by agarose gel electrophoresis.
2.5 specificity assay
The optimized PCR method is established by adopting the research, the primer P-1/P-2 is used for PCR amplification, and amplified samples are respectively pear-shaped worm positive samples, trypanosoma evanescens, unglued, hepatic clusterin, toxoplasma and Epimeria preserved in the laboratory, and agarose gel electrophoresis is carried out after the amplification.
2.6 sensitivity test
The extracted plasmid was measured for concentration using an ultra-micro spectrophotometer, 10 dilutions were diluted 10 times with the original concentration as the initial concentration, and PCR amplification was performed as template DNA, followed by agarose gel electrophoresis after amplification.
2.7 clinical sample detection
The blood of cattle and sheep collected in Guangxi Bai-shi city is extracted with DNA, and piroplasmosis detection and agarose gel electrophoresis are carried out by using the method established in the study. PCR detection and agarose gel electrophoresis were performed using primers designed by Casati, S.et al as controls, and were sent to Guangzhou Hua major Gene technologies Inc. for sequencing.
3. Results and analysis
3.1PCR amplification
Positive samples were amplified with the Piroparrangement primer (P-1/P-2) with a distinct specific band at 600bp, conforming to the expected size, as shown in FIG. 1.
Primers for Piroparrangement (P-1/P-2) amplified Hua Taile, theileria annulata, theileria euglena, babesia bisporus, babesia moellendorffii, babesia ovale and Babesia bovis all gave fragments of the expected 600bp size, as shown in FIG. 2.
3.2 optimization of optimum annealing temperature
The annealing temperature was optimized between 51℃and 69℃and the result showed that the band was brightest and the amplification was best at an annealing temperature of 61℃and therefore 61℃was chosen as the optimal annealing temperature as shown in FIG. 3.
3.3 specificity assay
The best conditions are used for respectively amplifying the piromo positive sample, the trypanosoma evanescens, the unglued, the liver cluster worm, the toxoplasma and the Ellison, and other bands are not found except the positive sample, wherein the band of the positive sample is 600bp and is the correct target band, and the result shows that the specificity of the established method of the experiment is good, as shown in figure 4.
3.4 sensitivity test
The plasmid concentration of Piroprion was determined to be 54.7 ng/. Mu.L by spectrophotometry. The PCR amplification is carried out after the plasmid sample is diluted by ten times, and the result shows that the minimum concentration which can be amplified by the Pirion PCR is 5.47 ag/. Mu.L, which indicates that the sensitivity of the PCR method established by the experiment is good, as shown in figure 5.
3.5 clinical sample detection
And carrying out PCR amplification on 24 clinical samples to detect 5 piroplasmosis positive, and comparing the detected piroplasmosis positive with a control detection result, wherein the sequencing result shows that the species of the piroplasmosis is infection is two Theileria orientalis and four Theileria luwenshuni, and the detection method established by the experiment can be used for clinical detection.
At present, the detection of Piriopsis cumingii in the prior art is mainly based on nested PCR, and is widely applied to the detection of various pathogens due to good specificity and high sensitivity. However, this method requires a long time and is not suitable for diagnosis of a large number of samples. The embodiment establishes a PCR method for detecting the piroplasmosis, and the time of the previous nest PCR method for detecting the piroplasmosis is halved. Through a specificity experiment, a sensitivity experiment and detection of clinical samples, the PCR method established by the embodiment has high sensitivity, good specificity and repeatability, and can be used for detection of clinical samples.
In addition, the PCR detection method established in the embodiment has obvious target bands when positive samples are detected, has no cross reaction with trypanosoma evanescens, no slurry, liver cluster, toxoplasma and Epimeria, has the minimum detection amount of 5.47 ng/mu L of piroplasmosis DNA, shows that the specificity is good, the sensitivity is high, the repeatability is high, and the PCR detection method can be used for clinical detection and epidemiological investigation.
The above specific embodiments are provided for illustrative purposes only and are not intended to limit the invention, and modifications, no inventive contribution, will be made to the embodiments by those skilled in the art after having read the present specification, as long as they are within the scope of the patent statutes.
Claims (3)
1. A PCR detection method for pirimiphos-type insects is characterized in that: the method comprises the following steps:
s1, sample collection: collecting 24 parts of blood of 24 cattle and sheep, respectively placing in anticoagulant tubes, and storing at-20deg.C;
s2, designing a primer and extracting whole blood DNA:
A. comparing the gene sequences of theileria inylanica 18S rRNA retrieved from NCBI by DNAStar7.1 software, and then designing universal primers P-1 and P-2 by adopting Premier6.0 software;
B. extracting the whole blood DNA of 24 parts of cattle and sheep blood in the step S1 by using a genome DNA purification kit, and storing the whole blood DNA at the temperature of-20 ℃ for later use;
s3, amplification of PCR:
1) PCR amplification is carried out by taking the DNA of the Theileria luwenshuni whole blood sample as a template and adopting a primer P-1/P-2, wherein the amplification system is as follows: 10 mu L of PCR premix, 6 mu L of dd H2O, 1 mu L of each of the upstream and downstream primers and 2 mu L of template DNA; the amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 61℃for 30s, extension at 72℃for 45s, and cycling for 35 times; extending at 72 ℃ for 8min;
2) Amplifying the Chinese theileria, the ring theileria, the Theileria ewingii, the babesia bifidus, the babesia moellendorffii, the babesia ovale and the babesia bovis respectively by using a primer P-1/P-2, detecting PCR products by using 1.2% agarose gel electrophoresis, and verifying whether the primer can amplify the Piroparrangement;
s4, constructing positive plasmids:
placing the agarose gel after electrophoresis in the step S3 in an ultraviolet analyzer, cutting off a target strip, placing the cut target strip in an EP tube with the volume of 1.5mL, then performing the operation of a DNA gel recovery kit, and finally measuring the concentration of a gel recovery product; the gel recovery product is connected with a pMD18-T carrier and then is transformed into DH-5 alpha competent cells, single bacterial colonies are selected through screening of ampicillin positive LB culture medium, and are subjected to shake cultivation for 3-5 hours at 37 ℃ and 200r/min, the obtained turbid bacterial liquid is used for extracting plasmids from bacterial liquid by using a kit, the concentration of the plasmids is measured by using a spectrophotometer, sequencing is carried out, whether the plasmids are target products or not is identified, and the residual plasmids are mixed with glycerol and then stored for standby; the plasmid identified as the product of interest was designated pMD-TH;
s5, extracting DNA from the collected cattle and sheep blood clinical samples according to the steps S1-S4, and detecting piroplasmosis and performing agarose gel electrophoresis.
2. The method for detecting the Pityrosporum tobrazii PCR according to claim 1, wherein the method comprises the following steps: the primer sequences in the step S2 are as follows:
P-1：5＇-CGGCTACCACATCTAAGGAAGG-3＇；
P-2：5＇-CAAGGTGCTGAAGGAGTCGTAA-3＇。
3. the method for detecting the Pityrosporum tobrazii PCR according to claim 1, wherein the method comprises the following steps: the gene sequences for alignment of the Theileria 18S rRNA gene sequences retrieved from NCBI in step S2 include Theileria orientalis MH208642.1, theileria annulata MF287948.1, theileria ewingii JF719835.1, theileria ovis MN493111.1, theileria reuteri LC326007.2, bulbia bifidus Bei Sichong MT539381.1, bulbia Bei Sichong KY805824.1, bulbeis bovis KP710223.1, and Bulbeis ovalis MN900525.1.
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