A yaks coronavirus detection kit based on a constant-temperature insulation fluorescence PCR platform and application

Abstract

The application discloses a kind of based on thermostatic insulation type fluorescent PCR platform's yak coronavirus detection kit and application, by reaction buffer, fluorescent quantitative PCR reaction liquid freeze-dried tube, positive control product and negative control product freeze-dried tube composition.Fluorescent quantitative PCR reaction liquid freeze-dried tube is mixed freeze-dried by Taq enzyme, reverse transcriptase, detection primer, probe and dNTPs.The kit of the application is short (reaction time is only 42 minutes) in detection time, strong specificity, high sensitivity, easy to store (4 ℃ is stored), cooperate thermostatic insulation type PCR instrument, very suitable for on-site rapid detection of yak coronavirus, can be widely popularized in grass-roots.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a yak coronavirus detection kit based on a constant-temperature isolation type fluorescent PCR platform and application. BACKGROUND

[0002] Bovine Coronavirus (BCV) was discovered in 1972 by Stair and Mebus et al. in the feces of diarrhea calves, proving to be one of the important pathogens causing diarrhea in calves (Jonsson E, Conway P. Probiotics for pigs [J]. Springer Netherlands, 1992.), and Yak BCV was first isolated in China in 1985 by Song Guanglin et al., but no further research was conducted. In 1990, Yao Huichun et al. found the existence of yak coronavirus in diarrhea of young yak, and indicated that yak coronavirus may be an important cause of yak diarrhea (Yao Huichun, Du Nianxing. Serological epidemiological investigation of bovine coronavirus [J]. Journal of Nanjing Agricultural University, 1990, 13(2): 117-121.). The clinical symptoms of yak infected with yak coronavirus are mainly diarrhea in newborn calves and diarrhea in adult cattle in winter, and the main clinical manifestations are hemorrhagic diarrhea in newborn calves and severe watery diarrhea in adult cattle, sometimes accompanied by blood and mucus, etc. which has brought huge economic losses to yak breeding industry.

[0003] There are many pathogens causing yak diarrhea, including rotavirus, viral diarrhea virus, Escherichia coli, Salmonella, Cryptosporidium and coccidia, etc., which are similar in symptoms and difficult to distinguish, so early differential diagnosis is particularly important, and pathogen detection can provide direct evidence of infection, which is the most reliable diagnostic method. PCR and fluorescent quantitative PCR are the most commonly used pathogen detection methods, and fluorescent quantitative PCR has the advantages of high sensitivity, strong specificity and good stability, and has become the mainstream method for animal disease diagnosis. However, the ordinary fluorescent quantitative PCR method is restricted in its application in on-site rapid detection due to the need for large equipment, complex operation and long reaction time. Therefore, it is urgent to establish a rapid, sensitive, specific and on-site detection method for yak coronavirus. SUMMARY

[0004] Therefore, the present application provides a yak coronavirus detection kit based on a constant-temperature isolation type fluorescent PCR platform and application, which is a simple, rapid, sensitive and specific detection method based on constant-temperature isolation type fluorescent PCR, and can provide scientific basis for on-site detection of yak coronavirus, which is of great significance to the healthy development of yak breeding industry.

[0005] In order to solve the above technical problems, the application discloses a primer and a probe for detecting yak coronavirus based on a constant-temperature insulation type fluorescent PCR platform, which comprises a yak coronavirus upstream primer, a yak coronavirus downstream primer and a yak coronavirus probe, wherein the nucleotide sequence of the yak coronavirus upstream primer is shown as SEQ ID NO. 1; the nucleotide sequence of the yak coronavirus downstream primer is shown as SEQ ID NO. 2; the nucleotide sequence of the yak coronavirus probe is shown as SEQ ID NO. 3; the 5' end of the nucleotide sequence of the yak coronavirus probe is connected with a FAM label, and the 3' end is connected with a non-fluorescent quenching group and a BHQ.

[0006] The application further discloses an application of the primer and the probe in preparation of a yak coronavirus detection kit.

[0007] The application further discloses a yak coronavirus detection kit based on the constant-temperature insulation type fluorescent PCR platform, which comprises a reaction buffer, a fluorescent quantitative PCR reaction liquid freeze-drying tube, a positive control product freeze-drying tube and a negative control product freeze-drying tube; the fluorescent quantitative PCR reaction liquid freeze-drying tube comprises 1.25 μL of Taq enzyme, 1.5 μL of reverse transcriptase, 3.0 μL of the yak coronavirus upstream primer, 3.0 μL of the yak coronavirus downstream primer and 0.3 μL of the yak coronavirus probe, and 3 μL of dNTPs.

[0008] Further, the final concentration of the Taq enzyme is 5 U·μL-1, the final concentration of the reverse transcriptase is 20 U·μL-1, the final concentration of the yak coronavirus upstream primer and the yak coronavirus downstream primer is 10 μmol·μL-1, the final concentration of the yak coronavirus probe is 10 μmol·μL-1, and the final concentration of the dNTPs is 2.5 mM.

[0009] Further, the fluorescent quantitative PCR reaction liquid freeze-drying tube is prepared by the following method: the above-mentioned Taq enzyme, reverse transcriptase, yak coronavirus upstream primer, yak coronavirus downstream primer and yak coronavirus probe and dNTPs are mixed and added into a 0.5 ml PCR tube, which is then reduced to-50 DEG C, and then freeze-dried for 24 h under a gas pressure of 10 pa to form a dry powder, and the tube is closed to obtain the fluorescent quantitative PCR reaction liquid freeze-drying tube.

[0010] Further, the fluorescent quantitative PCR reaction liquid freeze-drying tube is provided with 50 tubes.

[0011] Further, the reaction buffer is composed of the following components: 500 mM KCl, pH 8.3, 100 mM Tris-HCl, 15 mM MgCl2, and the rest is ddH2O, and the total volume of the above components is 3 ml.

[0012] Further, the positive control freeze-dried tube is prepared by the following method: 100 muL of RNA sample containing yak coronavirus RNA fragments is loaded into a PCR tube, and then frozen at-50 DEG C, and then freeze-dried at 10 pa for 24 h to form a dry powder, and then the tube is closed to prepare the positive control freeze-dried tube.

[0013] The negative control freeze-dried tube is prepared by the following method: 100 muL of sample without yak coronavirus RNA fragments is loaded into a PCR tube, and then frozen at-50 DEG C, and then freeze-dried at 10 pa for 24 h to form a dry powder, and then the tube is closed to prepare the negative control freeze-dried tube.

[0014] Further, the kit is stored at 4 DEG C.

[0015] The application further discloses a use method of the yak coronavirus detection kit based on the constant-temperature insulation type fluorescent PCR platform.

[0016] 1) Preparation of RNA template: 200 muL of supernatant of a fecal sample is taken, and total RNA of the detected sample supernatant is extracted according to the PetNAD kit extraction instruction of Jinruihongjie (Xiamen) Biological Technology Co., Ltd., and the RNA template is prepared.

[0017] 2) Preparation of the fluorescent PCR reaction system: the preparation of the fluorescent PCR reaction system is operated on ice; 100 muL of reaction buffer is added to the positive control freeze-dried tube to mix, and the positive control is prepared, and then 50 muL of reaction buffer and 5 muL of positive control are added to the fluorescent quantitative PCR reaction liquid freeze-dried tube to prepare the positive control reaction system; 100 muL of reaction buffer is added to the negative control freeze-dried tube to prepare the negative control, and then 50 muL of reaction buffer and 5 muL of negative control are added to the fluorescent quantitative PCR reaction liquid freeze-dried tube to prepare the negative control reaction system; 50 muL of reaction buffer and 5 muL of detected sample RNA are added to the fluorescent quantitative PCR reaction liquid freeze-dried tube to prepare the detected sample RNA reaction system; then 50 muL of mixture is taken from each reaction system and moved into the constant-temperature insulation type fluorescent PCR reaction tube; attention should be paid to light-proof operation when the sample is added; the prepared fluorescent quantitative PCR reaction tube is instantaneously and high-speed centrifuged for 5 s to avoid the generation of bubbles;

[0018] 3) Amplification detection: the PCR reaction tube is placed in the constant-temperature insulation type fluorescent PCR instrument, and the running key is pressed to start the reaction.

[0019] 4) Detection result determination: "+" represents positive, and "-" represents negative.

[0020] Compared with the prior art, the application can obtain the following technical effects:

[0021] 1) The application provides a fluorescent PCR detection kit for yak coronavirus, which has the advantages of high specificity, high sensitivity, short detection time, no pollution, no need for electrophoresis and on-site rapid detection.

[0022] 2) The application can quickly and accurately detect the yak coronavirus RNA in the detected sample, and can also detect the coronavirus RNA of ordinary cattle, and is used for molecular epidemiological investigation of cattle coronavirus.

[0023] 3) The reverse transcriptase and PCR amplification enzyme are included in the fluorescent quantitative PCR reaction solution of the application, so that the reverse transcription and amplification of nucleic acids are carried out in the same tube, the operation is simplified, the reagent is saved, and the possibility of RNA degradation and pollution is reduced.

[0024] 4) The fluorescent quantitative PCR reaction solution, positive and negative control products are prepared by freeze-drying, which reduces the storage conditions of the kit, can be stored at 4 DEG C for one year, is convenient for transportation and storage, and improves the stability of the reagent.

[0025] Of course, implementing any product of the application does not necessarily need to achieve all the technical effects described above. BRIEF DESCRIPTION OF DRAWINGS

[0026] The drawings described herein are used to provide further understanding of the application, and form a part of the application. The illustrative embodiments of the application and their descriptions serve to explain the application, and do not constitute an improper limitation on the application. In the drawings:

[0027] Figure 1 is a structure diagram of the POCKIT Micro instrument of the application;

[0028] Figure 2 is a positive sample sensitivity detection result of the application, wherein 1: 3.36x103 copies / μL; 2: 3.36x102 copies / μL; 3: 3.36x101 copies / μL; 4: 3.36x100 copies / μL. DETAILED DESCRIPTION

[0029] The embodiments of the application will be described in detail below with examples, so that the realization process of how the application applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented.

[0030] Example 1: Yak coronavirus detection kit based on constant temperature isolation type fluorescent PCR platform

[0031] A yak coronavirus detection kit based on a constant temperature isolation type fluorescent PCR platform comprises the following components:

[0032] (1) Reaction buffer: 3ml, consisting of 500mM KCl, 100mM Tris-HCl (pH 8.3), 15mM MgCl2 and ddH2O (balance).

[0033] (2) Fluorescent quantitative PCR reaction solution freeze-dried tube (50 tubes):

[0034] The fluorescent quantitative PCR reaction solution includes Taq enzyme 1.25 μL / tube (5 U·μL-1), reverse transcriptase 1.5 μL / tube (20 U·μL-1), detection upper and lower primers each 3.0 μL / tube (10 μmol·μL-1), and probe 0.3 μL / tube (10 μmol·μL-1), dNTPs 3 μL / tube (2.5 mM); the above mixture is added to a 0.5ml PCR tube, reduced to -50℃, then freeze-dried for 24h under 10pa pressure, and the dry powder is closed in a tube to prepare the fluorescent quantitative PCR reaction solution freeze-dried tube. Among them, the upper and lower primers and the probe for detecting the yak coronavirus are 3, which amplifies the length of 114p of the yak coronavirus polymerase gene, and the nucleotide sequence is shown in SEQ ID NO: 4.

[0035] Among them, the upper primer sequence is: 5'-AGTAGTGTCAGTGCTAAC-3', and the nucleotide sequence is shown in SEQ ID NO: 1; the lower primer sequence is: 5'-CAAGTGCCTGTAGGTATA-3'; and the nucleotide sequence is shown in SEQ ID NO: 2; the probe sequence of SEQ ID NO: 3 is: FAM-ACAACTTCCATCCCGCCAAA-BHQ1; and the nucleotide sequence is shown in SEQ ID NO: 3.

[0036] (3) Positive control freeze-dried tube (1 tube): 100 μL of RNA sample containing yak coronavirus RNA fragment is loaded into a PCR tube, reduced to -50℃, then freeze-dried for 24h under 10pa pressure, and the dry powder is closed in a tube to prepare.

[0037] (4) (4) Negative control freeze-dried tube (1 tube): 100 μL of sample without yak coronavirus RNA fragment is loaded into a PCR tube, reduced to -50℃, then freeze-dried for 24h under 10pa pressure, and the dry powder is closed in a tube to prepare.

[0038] The kit is stored at 4℃, and ice packs are required for transportation.

[0039] Example 2 Use method of yak coronavirus detection kit based on isothermal insulation fluorescent PCR platform

[0040] The method comprises the following steps:

[0041] 1) RNA template: 200 μL of fecal sample supernatant was taken, and the total RNA of the sample supernatant was extracted according to the extraction instructions of the PetNAD kit of Jinrui Hongjie (Xiamen) Biotechnology Co., Ltd., and the RNA template was prepared;

[0042] 2) Preparation of fluorescence PCR reaction system: the fluorescence PCR reaction system was prepared on ice;

[0043] 100 μL of reaction buffer was taken and added to the positive control freeze-dried tube for mixing to prepare the positive control, then 50 μL of reaction buffer and 5 μL of positive control were added to the fluorescence quantitative PCR reaction liquid freeze-dried tube to prepare the positive control reaction system; 100 μL of reaction buffer was added to the negative control freeze-dried tube to prepare the negative control, then 50 μL of reaction buffer and 5 μL of negative control were added to the fluorescence quantitative PCR reaction liquid freeze-dried tube to prepare the negative control reaction system; 50 μL of reaction buffer and 5 μL of detection sample RNA were added to the fluorescence quantitative PCR reaction liquid freeze-dried tube to prepare the detection sample RNA reaction system; then 50 μL of mixture was taken from each reaction system and transferred into a constant-temperature insulated fluorescence PCR reaction tube. When adding the sample, attention should be paid to avoid light operation. The prepared fluorescence quantitative PCR reaction tube was centrifuged at high speed for 5 s to avoid the formation of bubbles.

[0044] 3) Amplification detection: the PCR reaction tube was placed in a constant-temperature insulated fluorescence PCR instrument (POCKIT Micro) (as shown in Figure 1 ), and the running key was pressed to start the reaction;

[0045] 4) Detection result determination: "+" indicates positive, and "-" indicates negative.

[0046] Example 3 Preparation of standard and determination of sensitivity

[0047] 200 μL of fecal sample supernatant was taken, and the total RNA of the sample supernatant was extracted according to the extraction instructions of the PetNAD kit of Jinrui Hongjie (Xiamen) Biotechnology Co., Ltd., and then the total RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions of Baosheng Biotechnology (Dalian) Co., Ltd.

[0048] The prepared cDNA was subjected to PCR amplification with primers of the yak coronavirus, with the corresponding pathogenic cDNA as a positive control and no template as a negative control. The reaction system was as follows: 2.5 μL of cDNA, 12.5 μL of 2 × Taq PCR Master Mix, 1.0 μL of each of the upper and lower primers, 8.0 μL of ddH2O, and a total volume of 25 μL. The PCR amplification conditions were as follows: 5 min at 94 ℃, 30 s at 95 ℃, 30 s at 49 ℃, 30 s at 72 ℃, 35 cycles, and 10 min at 72 ℃. The PCR product was identified by 3% agarose gel electrophoresis, the target fragment was recovered with a gel recovery kit, and the fragment was cloned into a pMD19-T vector (Bao Bioengineering Co., Ltd.) and transformed into competent E. coli DH5α cells. The positive clones were selected and inoculated into LB liquid medium containing ampicillin, and cultured at 37 ℃ for 8 h. The recombinant plasmid was extracted with a plasmid extraction kit, and sent to Shengong Biotechnology Co., Ltd. for sequencing. The positive plasmid with correct sequencing was used as a positive standard of the yak coronavirus, and the concentration thereof was determined by a nucleic acid protein detector. The nucleic acid concentration was calculated according to the following formula: positive standard concentration (copies / μL) = (6.02 × 1023 copies / mol) × (100 ng / μl) × 10-9 / (1.791*106 g / mol) = 3.36 × 1010 copies / μL. The positive standard was diluted by 10 times in gradient (10-1-10-10), and the samples at four dilution degrees of 103, 102, 101 and 100 were detected. Each reaction tube was placed in a POCKIT Micro reaction hole, and the “run key” was pressed. The reaction was completed after 42 min. The positive result was “+”, and the negative result was “-”. The results are shown in Table 1. Figure 2 As shown in Table 1, the results of 103, 102 and 101 were “+”, and the result of 100 was “-”, indicating that the minimum detection limit of the kit was 33.6 copies / μL.

[0049] Example 4: Specificity and stability test

[0050] The kit developed in the present application was used to detect 22 positive samples of the yak coronavirus, 8 negative samples, and 10 related pathogens of the yak rotavirus, the yak viral diarrhea virus, the yak star virus, the yak enteritis virus, the yak E. coli, the yak Salmonella, the yak gas-producing capsular bacillus, the yak jejunal curved bacillus, the yak cryptosporidium, and the yak coccidia. The positive standard of the yak coronavirus was set up as a control to evaluate the specificity of the kit. The results showed that the detection of the 22 positive nucleic acid samples of the yak coronavirus was positive, the detection results of the 8 negative samples and the 10 related pathogens were negative, and the method had good specificity.

[0051] The kit is used for detecting 8 dilution positive standard samples of yak coronavirus for 3 times respectively, so as to evaluate the stability of the kit, and the detection results of the 3 times are consistent, and the stability of the kit is good.

[0052] Clinical application of the kit and comparison with common fluorescent quantitative PCR method

[0053] 30 total RNA samples extracted from yak coronavirus positive samples and 8 negative samples are detected by the kit and the fluorescent quantitative PCR method reported by Shen Fu (Shen Fu. Establishment and application of real-time fluorescent quantitative PCR detection method for bovine coronavirus [D]. Shandong Normal University, 2015.) to compare the coincidence rate of the two PCR methods.

[0054] 22 positive samples are detected by the kit, and the coincidence rate is 100%; while the fluorescent quantitative PCR method reported by Shen Fu detects 15 positive samples, and the coincidence rate is 68.2%. The detection results of the 8 negative samples are all negative, and the coincidence rate is 100%. The PCR product which is not detected by the fluorescent quantitative PCR method but detected by the kit is sequenced, and the sequencing result proves to be a specific sequence of the polymerase gene of yak coronavirus. It is proved that the kit is superior to the existing fluorescent quantitative PCR method in the detection effect of yak coronavirus, and is simpler and more portable than the existing PCR method, and is more suitable for on-site detection and diagnosis, thereby providing a powerful and convenient tool for the diagnosis and epidemiological investigation of yak coronavirus.

[0055] The kit is used in cooperation with the POCKIT Micro instrument, is simple and portable, is suitable for clinical detection, and is not commercially available at present.

[0056] The reaction reagent is freeze-dried in the kit, so that the storage condition of the kit is reduced, and the transportation and clinical application are facilitated.

[0057] Due to the harsh natural environment of the Qinghai-Tibet Plateau, some pathogens have been mutated, so that the detection rate of some PCR methods is reduced. The primers and probes of the kit are designed according to the conserved sequences in the mutated strains and unmutated strains on the Qinghai-Tibet Plateau, so that the detection rate of the method is greatly improved, and therefore the method is superior to the method reported in the literature.

[0058] The foregoing description illustrates and describes the only preferred embodiments of this invention, and is not intended to be exhaustive or to necessarily limit the invention to the precise form disclosed. Many modifications and variations are possible in light of this teaching. It is intended that the scope of the invention be limited not with this detailed description, but rather by the claims appended hereto. Any reference to prior art contained herein is for illustrative purposes, and is intended to serve only as an example of toolsconveniently available to assist with understanding this invention and the manner in which it can be made and utilized. However, alternative examples of tools, both known and unknown, can be readily implemented to practice the present invention. Any implementation of the present invention can be made using tools that differ from those expressly described herein, and it is intended that the present invention not be limited to the tools expressly described herein. SEQUENCE LISTING <110> Southwestern University for Nationalities <120> A yak coronavirus detection kit based on a constant-temperature insulation fluorescence PCR platform and application <130> 2017 <141> 2017-12-22 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial sequence <400> 1 agtagtgtca gtgctaac 18 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 caagtgcctg taggtata 18 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 acaacttcca tcccgccaaa 20 <210> 4 <211> 114 <212> DNA <213> Bovine Coronavirus <400> 4 AGTAGTGTCA GTGCTAACAC ATGAACAACT TCCATCCCgc caaaatccac aaacttgaca 60 AACATCATGT GTCAAACAT AAGACACAGG ATCTTTTATA CCTACAGGCA TTG 114

Claims

1. A detection kit for bovine coronavirus based on a thermostatic insulation fluorescence PCR platform, characterized in that, The reaction buffer, the freeze-dried tube of the fluorescent quantitative PCR reaction solution, the freeze-dried tube of the positive control product and the freeze-dried tube of the negative control product are included. The freeze-dried tube of the fluorescent quantitative PCR reaction solution is prepared by the following method: 1.25 μL of Taq enzyme, 1.5 μL of reverse transcriptase, 3.0 μL of the upstream primer of the yak coronavirus, 3.0 μL of the downstream primer of the yak coronavirus, 0.3 μL of the yak coronavirus probe and 3 μL of dNTPs are mixed and added into a 0.5 ml PCR tube, which is then reduced to-50℃, and then freeze-dried for 24 h under 10 pa of air pressure to form a dry powder, and the tube is closed to obtain the freeze-dried tube of the fluorescent quantitative PCR reaction solution. wherein the final concentration of the Taq enzyme is 5 U per μL -1 , the final concentration of the reverse transcriptase is 20 U per μL -1 , the final concentration of the yak coronavirus upstream primer and the yak coronavirus downstream primer is 10 μmol per μL -1 , the final concentration of the yak coronavirus probe is 10 μmol per μL -1 , the final concentration of the dNTPs is 2.5 mM; The nucleotide sequence of the upstream primer of the yak coronavirus is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer of the yak coronavirus is shown as SEQ ID NO. 2; the nucleotide sequence of the yak coronavirus probe is shown as SEQ ID NO. 3; the 5 end of the nucleotide sequence of the yak coronavirus probe is connected with a FAM label, and the 3 end is connected with a BHQ.

2. The Yunnan golden monkey coronavirus detection kit based on the thermostatic insulation fluorescence PCR platform according to claim 1, characterized in that, The freeze-dried tube of the fluorescent quantitative PCR reaction solution is provided with 50 tubes.

3. The Yunnan golden monkey coronavirus detection kit based on the thermostatic insulation fluorescence PCR platform according to claim 1, characterized in that, The reaction buffer is composed of the following components: 500 mM KCI, 100 mM Tris-HCI with pH of 8.3, 15 mM MgCl2, and the rest is ddH2O, and the total volume of the reaction buffer is 3 ml.

4. The Yunnan golden monkey coronavirus detection kit based on the thermostatic insulation fluorescence PCR platform according to claim 1, characterized in that, The freeze-dried tube of the positive control product is prepared by the following method: 100 μL of an RNA sample containing a yak coronavirus RNA fragment is loaded into a PCR tube, which is then reduced to-50℃, and then freeze-dried for 24 h under 10 pa of air pressure to form a dry powder, and the tube is closed to obtain the freeze-dried tube of the positive control product. The freeze-dried tube of the negative control product is prepared by the following method: 100 μL of a sample without a yak coronavirus RNA fragment is loaded into a PCR tube, which is then reduced to-50℃, and then freeze-dried for 24 h under 10 pa of air pressure to form a dry powder, and the tube is closed to obtain the freeze-dried tube of the negative control product.

5. The Yunnan golden monkey coronavirus detection kit based on the thermostatic insulation fluorescence PCR platform according to claim 1, characterized in that, The kit is stored at 4℃.

Images