CN116200543A - Method, primer composition and kit for rapid detection of feline-canine pathogens - Google Patents
Method, primer composition and kit for rapid detection of feline-canine pathogens Download PDFInfo
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Abstract
The invention relates to a method, a primer composition and a kit for rapidly detecting a cat and dog pathogen, which are used for solving the technical problems of low detection accuracy, high cost, complex detection process, long time, very high equipment and equipment cost and the like of the existing pet pathogen detection method. The method comprises the steps of 1, collecting pathogen samples to be detected; 2. subjecting the sample to nucleic acid purification or direct lysis treatment by a lysis solution; 3. an amplification reaction; 3.1, determining a specific primer group for each pathogen to be detected and designing an amplification system; 3.2, adding the sample treated in the step 2 and the corresponding primer group into an amplification system for amplification reaction; 4. visual detection; and observing whether the color of the reaction solution after the amplification reaction changes, if the color changes, infecting the corresponding pathogen, and if the color does not change, not infecting the corresponding pathogen.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a method for rapidly detecting a feline-canine pathogen, a primer composition and a kit.
Background
With the development of society and the improvement of economic level, the households for raising pets are increasing. Compared to humans, pets live in a more rough environment and diet, making them more susceptible to infection by pathogens. Early diagnosis of the infected pathogens of pets helps to take therapeutic measures in time to restore health and prevent cross infection of pathogens in humans.
The detection method of pet pathogens in the current market mainly depends on a colloidal gold immunochromatography method and a real-time fluorescence PCR method. The colloidal gold immunochromatography is simple and quick to operate and free of equipment dependence, so that the market share is high. However, the detection accuracy of the colloidal gold immunochromatography is very low, only about 60%, and the preparation process of the immunochromatography test strip requires antibody coating to functionalize the colloidal gold so as to greatly increase the detection cost. The accuracy of the real-time fluorescence PCR method is high, but the method is very high in equipment and equipment cost, the operation of the detection process is complex, and the corresponding requirements are also met on the experimental space of the detection environment, so that the method is difficult to bear for common pet hospitals and carry out the detection of the real-time fluorescence PCR method. In addition, since the collected sample contains various DNA polymerase inhibitors, such as hemoglobin in whole blood samples, humic acid in fecal samples, protease in nasopharyngeal swabs, etc., can inhibit nucleic acid amplification, the pet pathogen must be purified from nucleic acid before detection to remove the inhibitors for subsequent amplification, which increases not only the detection steps and time, but also the risk of cross contamination.
In order to meet the increasingly huge domestic pet pathogen detection market and meet the pathogen detection requirements of common pet hospitals, it is necessary to invent a simple, quick and low-cost pet pathogen detection method which does not depend on instruments and equipment.
Disclosure of Invention
The invention aims to provide a rapid detection method, a primer composition and a kit for cat and dog pathogens, which are used for solving the technical problems of low detection accuracy, high cost, complex detection process, long time, very high equipment and equipment cost and the like of the existing pet pathogen detection method.
The technical scheme of the invention is to provide a method for rapidly detecting a cat and dog pathogen, which is characterized by comprising the following steps:
step 1, collecting pathogen samples to be detected;
step 2, sample processing;
the nucleic acid purification method is adopted, and the pathogen nucleic acid is extracted from the sample collected in the step 1 through nucleic acid purification; or, directly cracking the sample acquired in the step 1 by using a cracking liquid;
step 3, amplification reaction;
step 3.1, determining specific primer groups aiming at each pathogen to be detected, wherein the primer groups together form a primer composition for rapid detection of the cat and dog pathogens;
meanwhile, an amplification system is designed, which comprises:
1mM-80mM Tris-HCl, 10mM-500mM KCl, 0.2mM-30mM MgSO 4 MgCl of 10mM-500mM 2 Tween-20 at a concentration of 0.001-20% by volume, 0.5mM-120mM (NH) 4 ) 2 SO 4 0.1M-15M betaine, 0.2mM-5mM dNTP, 1-40U DNA polymerase, 0.1-20U UDG enzyme, 0.01pM-50M pH indicator and DNA polymerase enhancer;
step 3.2, adding the sample treated in the step 2 and the corresponding primer group into an amplification system for amplification reaction;
step 4, visual detection;
observing whether the color of the reaction solution changes after the amplification reaction, and judging whether the detected sample is infected with a corresponding pathogen; if there is a color change, the corresponding pathogen is infected, and if there is no color change, the corresponding pathogen is not infected.
Further, the DNA polymerase enhancer is one or two of gelatin and 15mM-100mM trimethylamine oxide with the mass volume ratio of 0.1% -10%.
Further, the dntps are dATP, dTTP, dCTP, dGTP and dUTP.
Further, the UDG enzyme is uracil-DNA glycosylase.
Further, the pH indicator is bromocresol purple, naphtholphthalein, thymol blue, m-cresol purple, phenol red, cresol red or xylenol orange, and combinations of any two or more thereof.
Further, step 3.2 uses isothermal amplification technique to achieve amplification. Compared with other nucleic acid isothermal amplification technologies, the loop-mediated isothermal amplification technology has higher amplification efficiency, can generate a large amount of hydrogen ions in the amplification stage to change the pH value of an amplification system, and is insensitive to the change of the pH value in a certain range, so that the invention adopts the technology to amplify target genes.
Further, the lysate in step 2 is 1mM-30M inorganic salt solution or 0.001% -90% organic solution and combinations thereof.
Further, the inorganic salt solution is NaCl solution, liCl solution, KCl solution, naOH solution or KOH solution; the organic solution is SDS solution, EDTA solution, triton X-100 solution, tween-20 solution, tween-80 solution or PEG-200 solution.
Further, the pathogen sample to be detected in the step 1 is body fluid, tissue fluid, secretion fluid of the individual cat and dog to be detected, a nasopharyngeal swab, an oral swab or an anal swab.
Further, the primer set for determining the specificity for each pathogen to be detected in step 3.1 is specifically as follows:
the pathogen to be detected is cat parvovirus, and the specific primer group nucleic acid sequence is shown as SEQ ID NO 1-SEQ ID NO 4, specifically:
upstream outer primer: TGTTTAAACTTGGGCGGG
Downstream outer primer: ATAGTTGTTCCAGCGAACAT
Upstream inner primer: GACAAGAAGCGAATGTTTGTGGGCGTGGTTAAAG
Downstream inner primer: AGAGTGAACCTCTCTTTCCTCAGTATACTGGTT
The pathogen to be detected is canine parvovirus, and the specific primer group nucleic acid sequence is shown in SEQ ID NO 5-SEQ ID NO 8, specifically:
upstream outer primer: AAACTAAAAAGGAAGTGTCA
Downstream outer primer: GTATTAATTCAAATGCT
Upstream inner primer: AACTATCTGGTTGTTGCGGGACTTGGTTAGTAAAAG
Downstream inner primer: GATGGCACAACCAGGAAAAGTCAAAGTACAAATTTC
The pathogen to be detected is feline herpesvirus, the specific primer group nucleic acid sequence is shown in SEQ ID NO 9-SEQ ID NO 12, and specifically:
upstream outer primer: ATGTCTTTTTGAAGTCT
Downstream outer primer: TCACCCATTCACTATCTT
Upstream inner primer: CAGCCTTGATCTTTTCCAGAACCAAATGAATGT
Downstream inner primer: ATGTTTTACTTCATATTTGTCTGCGTAGCC.
Further, the pathogen nucleic acid extracted in step 2 includes DNA and RNA; wherein the DNA comprises genomic DNA, episomal DNA and pathogen DNA of the detected individual, and the RNA comprises total RNA, episomal RNA and pathogen RNA of the detected individual.
Further, the pH of the amplification system is between 2 and 13.
The invention also provides a primer composition for rapidly detecting the cat and dog pathogens, which is characterized in that: comprises at least one group of a feline parvovirus primer group, a canine parvovirus primer group and a feline herpesvirus primer group;
the nucleic acid sequences of the cat parvovirus primer group are shown in SEQ ID NO 1-SEQ ID NO 4, and are respectively:
upstream outer primer: TGTTTAAACTTGGGCGGG
Downstream outer primer: ATAGTTGTTCCAGCGAACAT
Upstream inner primer: GACAAGAAGCGAATGTTTGTGGGCGTGGTTAAAG
Downstream inner primer: AGAGTGAACCTCTCTTTCCTCAGTATACTGGTT
The nucleic acid sequences of the canine parvovirus primer group are shown in SEQ ID NO 5-SEQ ID NO 8, and are respectively:
upstream outer primer: AAACTAAAAAGGAAGTGTCA
Downstream outer primer: GTATTAATTCAAATGCT
Upstream inner primer: AACTATCTGGTTGTTGCGGGACTTGGTTAGTAAAAG
Downstream inner primer: GATGGCACAACCAGGAAAAGTCAAAGTACAAATTTC
The nucleic acid sequences of the feline herpesvirus primer group are shown in SEQ ID NO 9-SEQ ID NO 12, respectively:
upstream outer primer: ATGTCTTTTTGAAGTCT
Downstream outer primer: TCACCCATTCACTATCTT
Upstream inner primer: CAGCCTTGATCTTTTCCAGAACCAAATGAATGT
Downstream inner primer: ATGTTTTACTTCATATTTGTCTGCGTAGCC.
The invention also provides a kit for rapidly detecting the cat and dog pathogens, which is characterized in that: a primer composition comprising the primer set of claim 15 for rapid detection of a feline canine pathogen.
Further, the kit further comprises an amplification system, wherein the amplification system comprises 1mM-80mM Tris-HCl, 10mM-500mM KCl, 0.2mM-30mM MgSO4, 10mM-500mM MgCl2, tween-20 with a volume concentration of 0.001% -20%, 0.5mM-120mM (NH) 4 ) 2 SO 4 0.1M-15M betaine, 0.2mM-5mM dNTP, 1-40U DNA polymerase, 0.1-20U UDG enzyme, 0.01pM-50M pH indicator and DNA polymerase enhancer.
Further, the DNA polymerase enhancer is one or two of gelatin and 15mM-100mM trimethylamine oxide with the mass volume ratio of 0.1% -10%.
Further, the dntps are dATP, dTTP, dCTP, dGTP and dUTP.
Further, the UDG enzyme is uracil-DNA glycosylase.
Further, the pH indicator is bromocresol purple, naphtholphthalein, thymol blue, m-cresol purple, phenol red, cresol red or xylenol orange, and combinations thereof.
The beneficial effects of the invention are as follows:
(1) The detection process is simple and convenient;
the existing pathogen detection method based on nucleic acid amplification requires nucleic acid purification to obtain a high-purity detection template during nucleic acid amplification, which causes the increase of detection cost, tedious operation and prolonged detection period. According to the invention, by optimizing an amplification system and using Gelatin (Gelatin) or TMAO (trimethylamine oxide) as an enhancer of DNA polymerase, a cracked sample can be directly used for amplification, so that the process of nucleic acid purification is avoided, the operation steps are simplified, the detection time is shortened, and the risk of cross contamination between samples in the process of nucleic acid purification is reduced.
The Gelatin (Gelatin) or TMAO (trimethylamine oxide) is used as the enhancer of DNA polymerase, so that the activity of the DNA polymerase can be enhanced during nucleic acid amplification, the adsorption of the DNA polymerase on the tube wall is reduced, the tolerance strength of an amplification system to the DNA polymerase inhibitor in a sample is integrally enhanced, and therefore, the nucleic acid amplification can be successfully completed under the condition that the DNA polymerase inhibitor exists for the sample which is not subjected to nucleic acid purification only by simple cleavage.
(2) The detection cost is low, and the popularization and the use are convenient;
existing pet pathogen detection technologies, such as colloidal gold immunochromatography and real-time fluorescence PCR, require antibodies to modify colloidal gold or rely on complex, precise and expensive instruments and equipment, so that the output of detection results requires high cost. The detection method provided by the invention is not dependent on complex instruments while accurately detecting, and can visually judge the result only by naked eyes, so that the detection cost is greatly reduced, and the method is convenient to popularize and use in various pet hospitals.
(3) The detection efficiency is high;
existing pathogen detection methods based on nucleic acid amplification rely on PCR technology, which relies on thermal cycling to amplify the entire nucleic acid for up to 90 minutes. According to the invention, the nucleic acid isothermal amplification technology is adopted without nucleic acid purification, so that the thermal cycle is not needed, the nucleic acid amplification process can be finished in an isothermal environment for 30min, and the detection efficiency is greatly improved.
(4) The detection sensitivity and the specificity are high;
compared with the existing pathogen detection method based on the real-time fluorescence PCR method, the nucleic acid isothermal amplification technology adopted by the invention has the advantages that the accuracy and the operation simplicity are ensured, and meanwhile, the high sensitivity and the high specificity are obtained through optimizing the primer design, optimizing the reaction system and other conditions. Trace amounts of pathogen nucleic acid can be detected early in the pathogen infection.
(5) Preventing aerosol pollution;
the aerosol generated in the nucleic acid amplification process can exist in the detection environment for a long time, and the accuracy of the detection result is seriously interfered. The existing pathogen detection method based on nucleic acid amplification does not introduce an anti-pollution system in the method design, and the accuracy of detection results is greatly reduced. The invention adopts an anti-pollution system based on UDG enzyme, thereby effectively avoiding the interference of aerosol on detection results.
(6) The application range is wide;
the amplification system is also suitable for samples subjected to nucleic acid purification, and compared with the existing amplification system, the amplification system provided by the invention adopts a nucleic acid isothermal amplification technology with higher amplification efficiency, and can finish amplification in a shorter time.
Drawings
FIG. 1 is a graphical representation of the color signals of the negative and positive pathogen detection results in the examples (wherein the actual color development in the a-tube is orange and the actual color development in the b-tube is rose).
Detailed Description
So that the manner in which the above recited objects, features and advantages of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to the embodiments, some of which are illustrated in the appended drawings. All other embodiments, which can be made by one of ordinary skill 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.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
The invention discloses a rapid detection method, a primer composition and a kit for a cat and dog pathogen. The specific method comprises the following steps: collecting a sample, processing the sample, amplifying and visually detecting; the sample type is blood, saliva, tear tissue fluid, secretion fluid or body fluid, nasopharyngeal swab, buccal swab, anal swab, etc. Through optimizing the amplification system, the method can be used for nucleic acid amplification of pathogens without nucleic acid purification and direct cleavage by the lysate in a sample processing link, and meanwhile, the pH value of a specially designed amplification system is changed during nucleic acid amplification, so that macroscopic color change is generated to indicate whether nucleic acid amplification occurs or not. Of course, the amplification system is also suitable for nucleic acid purified samples; types of nucleic acids extracted by nucleic acid purification include DNA and RNA. Wherein the DNA comprises genomic DNA, episomal DNA and pathogen DNA of the detected individual, and the RNA comprises total RNA, episomal RNA and pathogen RNA of the detected individual.
The amplification system used in the present invention comprises:
1mM-80mM Tris-HCl, 10mM-500mM KCl, 0.2mM-30mM MgSO 4 MgCl of 10mM-500mM 2 The concentration of Tween-20 with the volume concentration of 0.001-20%, dNTP with the volume concentration of 0.2-5 mM, DNA polymerase with the volume of 1-40U, UDG enzyme with the volume of 0.1-20U, pH indicator with the volume of 0.01pM-50M and DNA polymerase enhancer, wherein the content of each component in an amplification system refers to the concentration of the corresponding component in the amplification system, and the content of Tris-HCl in a 1L amplification system solution is 1-80mmol by taking Tris-HCl as an example. Wherein the DNA polymerase enhancer is Gelatin (Gelatin) with the mass volume ratio of 0.1-10%, 15mM-100mM TMAO (trimethylamine oxide) or the combination of the two; dNTPs are dATP, dTTP, dCTP, dGTP and dUTP; the UDG enzyme is uracil-DNA glycosylase; the pH indicator is bromocresol purple, naphtholphthalein, thymol blue, m-cresol purple, phenol red, cresol red or xylenol orange, and combinations thereof. The pH of the amplification system is between 5 and 10.
In addition, the primer design and the amplification reaction conditions can be optimized, the accuracy and the operation simplicity are ensured, meanwhile, the high sensitivity and the high specificity are obtained, meanwhile, the pathogen-specific nucleic acid fragments can be greatly amplified in a short time under the constant temperature condition, and trace pathogen nucleic acid can be detected at the early stage of pathogen infection.
The present invention designs the following primer compositions for different pathogens:
the pathogen to be detected is cat parvovirus, and the specific primer group nucleic acid sequence is shown as SEQ ID NO 1-SEQ ID NO 4, specifically:
upstream outer primer: TGTTTAAACTTGGGCGGG
Downstream outer primer: ATAGTTGTTCCAGCGAACAT
Upstream inner primer: GACAAGAAGCGAATGTTTGTGGGCGTGGTTAAAG
Downstream inner primer: AGAGTGAACCTCTCTTTCCTCAGTATACTGGTT
The pathogen to be detected is canine parvovirus, and the specific primer group nucleic acid sequence is shown in SEQ ID NO 5-SEQ ID NO 8, specifically:
upstream outer primer: AAACTAAAAAGGAAGTGTCA
Downstream outer primer: GTATTAATTCAAATGCT
Upstream inner primer: AACTATCTGGTTGTTGCGGGACTTGGTTAGTAAAAG
Downstream inner primer: GATGGCACAACCAGGAAAAGTCAAAGTACAAATTTC
The pathogen to be detected is feline herpesvirus, the specific primer group nucleic acid sequence is shown in SEQ ID NO 9-SEQ ID NO 12, and specifically:
upstream outer primer: ATGTCTTTTTGAAGTCT
Downstream outer primer: TCACCCATTCACTATCTT
Upstream inner primer: CAGCCTTGATCTTTTCCAGAACCAAATGAATGT
Downstream inner primer: ATGTTTTACTTCATATTTGTCTGCGTAGCC.
The invention also provides a rapid detection kit for the feline-canine pathogens, which comprises the primer composition and the amplification system.
The detection method of the present invention will be described in detail with reference to specific examples.
Example 1
This example illustrates the detection of feline parvovirus:
the present example is directed to the design of specific primer sets for feline parvovirus genes: the gene sequence of feline parvovirus was confirmed by NCBI database, and synthetic primers were designed accordingly. To increase amplification specificity and detection resolution, multiple sets of primers were designed for assay screening.
Screening the optimal primers according to the following targets: (1) high amplification specificity: only samples infected with feline parvovirus initiate nucleic acid amplification and result in significant signal generation. (2) The amplification sensitivity is high, and fragments of interest as low as 100copies can be detected. (3) The method has high adaptability to sample types, is suitable for high-purity templates subjected to nucleic acid purification, and can effectively initiate nucleic acid amplification for rough samples without nucleic acid purification.
The final designed primer group is shown as SEQ ID NO 1-SEQ ID NO 4, and specifically comprises the following steps:
upstream outer primer: TGTTTAAACTTGGGCGGG
Downstream outer primer: ATAGTTGTTCCAGCGAACAT
Upstream inner primer: GACAAGAAGCGAATGTTTGTGGGCGTGGTTAAAG
Downstream inner primer: AGAGTGAACCTCTCTTTCCTCAGTATACTGGTT
Anal swab samples were taken from 10 cats infected with feline parvovirus, diagnosed by real-time fluorescence PCR, and two anal swabs were taken from each affected cat. One of the swabs is subjected to commercial nucleic acid purification kits to extract viral nucleic acids for nucleic acid amplification; the other swab is directly cracked by the lysate for nucleic acid amplification, and the lysate is as follows: 1mM-30M inorganic salt solution or 0.001% -90% organic solution and combinations thereof. The inorganic salt solution is NaCl solution, liCl solution, KCl solution, naOH solution or KOH solution; the organic solution is SDS solution, EDTA solution, triton X-100 solution, tween-20 solution, tween-80 solution or PEG-200 solution. In specific lysis, the anal swab was immersed in a 1.5mL centrifuge tube containing 500 μl of lysis solution, incubated at room temperature for 1min, the swab was discarded, the obtained solution was transiently centrifuged at room temperature for 3min, and 1 μl of supernatant was used for nucleic acid amplification.
The following amplification systems were placed in 200 μl centrifuge tubes:
placing each reaction component and a sample to be detected into a centrifuge tube, wherein the pH is 8.8, the whole body is pink, and placing into a metal bath for incubation for 30min at the constant temperature of 60 ℃.
After incubation, judging the detection result according to the following principle: as shown in FIG. 1, if the color of the reaction system in the centrifuge tube has obvious chromatic aberration compared with that before the reaction, the reaction system is judged to be positive; if the color of the reaction system in the centrifuge tube has no obvious color difference compared with the color before the reaction, the reaction system is judged to be negative. Compared with the conventional detection method requiring nucleic acid purification, the accuracy of the present embodiment is completely consistent, but the detection time is greatly shortened and the operation steps are simplified.
Example 2 detection of canine parvovirus
The present example illustrates canine parvovirus detection:
designing a specific primer group aiming at canine parvovirus genes: the gene sequence of canine parvovirus was confirmed by NCBI database, and synthetic primers were designed accordingly. To increase amplification specificity and detection resolution, multiple sets of primers were designed for assay screening.
Screening the optimal primers according to the following targets: (1) high amplification specificity: only samples infected with canine parvovirus initiate nucleic acid amplification and result in significant signal generation. (2) The amplification sensitivity is high, and fragments of interest as low as 100copies can be detected. (3) The method has high adaptability to sample types, is suitable for high-purity templates subjected to nucleic acid purification, and can effectively initiate nucleic acid amplification for rough samples without nucleic acid purification.
The final designed primer group is shown as SEQ ID NO 5-SEQ ID NO 8, and specifically comprises the following steps:
upstream outer primer: AAACTAAAAAGGAAGTGTCA
Downstream outer primer: GTATTAATTCAAATGCT
Upstream inner primer: AACTATCTGGTTGTTGCGGGACTTGGTTAGTAAAAG
Downstream inner primer: GATGGCACAACCAGGAAAAGTCAAAGTACAAATTTC
Anal swab samples were taken from 10 dogs infected with canine parvovirus confirmed by a real-time fluorescent PCR method, and two anal swabs were taken from each affected dog. One of the swabs is subjected to commercial nucleic acid purification kits to extract viral nucleic acids for nucleic acid amplification; the other swab is directly cracked by the lysate of the invention for nucleic acid amplification, specifically, the anal swab is immersed in a 1.5mL centrifuge tube filled with 500 mu L of lysate, the swab is discarded after incubation for 1min at room temperature, the obtained solution is instantaneously centrifuged for 3min at room temperature, and 1 mu L of supernatant is taken for nucleic acid amplification.
The following amplification systems were placed in 200 μl centrifuge tubes:
the centrifuge tube was placed in a metal bath and incubated at 60℃for 30min.
After incubation, judging the detection result according to the following principle: as shown in FIG. 1, if the color of the reaction system in the centrifuge tube has obvious chromatic aberration compared with that before the reaction, the reaction system is judged to be positive; if the color of the reaction system in the centrifuge tube has no obvious color difference compared with the color before the reaction, the reaction system is judged to be negative.
Example 3 detection of feline herpesvirus
This example illustrates the detection of feline herpes virus:
specific primer groups are designed for the feline herpesvirus genes: the gene sequence of feline herpesvirus was confirmed by NCBI database, and synthetic primers were designed accordingly. To increase amplification specificity and detection resolution, multiple sets of primers were designed for assay screening.
Screening the optimal primers according to the following targets: (1) high amplification specificity: only samples infected with feline herpesvirus initiate nucleic acid amplification and result in a significant signal generation. (2) The amplification sensitivity is high, and fragments of interest as low as 100copies can be detected. (3) The method has high adaptability to sample types, is suitable for high-purity templates subjected to nucleic acid purification, and can effectively initiate nucleic acid amplification for rough samples without nucleic acid purification.
The final designed primer group is shown as SEQ ID NO 9-SEQ ID NO 12, and specifically comprises the following steps:
upstream outer primer: ATGTCTTTTTGAAGTCT
Downstream outer primer: TCACCCATTCACTATCTT
Upstream inner primer: CAGCCTTGATCTTTTCCAGAACCAAATGAATGT
Downstream inner primer: ATGTTTTACTTCATATTTGTCTGCGTAGCC.
Nasopharyngeal swab samples were taken from 10 cats infected with feline herpesvirus confirmed by real-time fluorescent PCR, and two nasopharyngeal swabs were taken per cat. One of the swabs is subjected to commercial nucleic acid purification kits to extract viral nucleic acids for nucleic acid amplification; the other swab is directly lysed by the lysate of the invention for nucleic acid amplification, specifically, the nasopharyngeal swab is immersed in a 1.5mL centrifuge tube filled with 500 mu L of the lysate, the swab is discarded after incubation for 1min at room temperature, the obtained solution is instantaneously centrifuged for 3min at room temperature, and 1 mu L of the supernatant is taken for nucleic acid amplification.
The following amplification systems were placed in 200 μl centrifuge tubes:
the centrifuge tube was placed in a metal bath and incubated at 60℃for 30min.
After incubation, judging the detection result according to the following principle: if the color of the reaction system in the centrifuge tube has obvious chromatic aberration compared with that before the reaction, judging that the reaction system is positive; if the color of the reaction system in the centrifuge tube has no obvious color difference compared with the color before the reaction, the reaction system is judged to be negative.
Claims (19)
1. A method for rapid detection of a feline-canine pathogen, comprising the steps of:
step 1, collecting pathogen samples to be detected;
step 2, sample processing;
the nucleic acid purification method is adopted, and the pathogen nucleic acid is extracted from the sample collected in the step 1 through nucleic acid purification; or, directly cracking the sample acquired in the step 1 by using a cracking liquid;
step 3, amplification reaction;
step 3.1, determining specific primer groups aiming at each pathogen to be detected, wherein the primer groups together form a primer composition for rapid detection of the cat and dog pathogens;
simultaneously designing an amplification system, wherein the amplification system comprises:
1mM-80mM Tris-HCl, 10mM-500mM KCl, 0.2mM-30mM MgSO 4 MgCl of 10mM-500mM 2 Tween-20 at a concentration of 0.001-20% by volume, 0.5mM-120mM (NH) 4 ) 2 SO 4 0.1M-15M betaine, 0.2mM-5mM dNTP, 1-40U DNA polymerase, 0.1-20U UDG enzyme, 0.01pM-50M pH indicator and DNA polymerase enhancer;
step 3.2, adding the sample treated in the step 2 and the corresponding primer group into an amplification system for amplification reaction;
step 4, visual detection;
and (3) observing whether the color of the reaction solution changes after the amplification reaction, judging whether the detected sample is infected by a corresponding pathogen, if the color changes, the detected sample is infected by the corresponding pathogen, and if the color changes, the detected sample is not infected by the corresponding pathogen.
2. The method for rapid detection of feline canine pathogens according to claim 1, wherein: the DNA polymerase enhancer is one or two of gelatin and trimethylamine oxide with the mass volume ratio of 0.1-10% and 15-100 mM.
3. The method for rapid detection of feline canine pathogens according to claim 2, wherein: the dNTPs are dATP, dTTP, dCTP, dGTP and dUTP.
4. A method for rapid detection of a feline canine pathogen according to claim 3 wherein: the UDG enzyme is uracil-DNA glycosylase.
5. The method for rapid detection of a feline canine pathogen according to claim 4, wherein: the pH indicator is bromocresol purple, naphtholphthalein, thymol blue, m-cresol purple, phenol red, cresol red or xylenol orange, or a combination of any two or more thereof.
6. The method for rapid detection of a feline canine pathogen according to claim 4, wherein: and 3.2, realizing amplification by adopting a constant-temperature amplification technology.
7. The method for rapid detection of a feline-canine pathogen according to any one of claims 1-6, wherein: the lysate in step 2 is 1mM-30M inorganic salt solution or 0.001% -90% organic solution and the combination thereof.
8. The method for rapid detection of a feline canine pathogen according to claim 7, wherein: the inorganic salt solution is NaCl solution, liCl solution, KCl solution, naOH solution or KOH solution; the organic solution is SDS solution, EDTA solution, triton X-100 solution, tween-20 solution, tween-80 solution or PEG-200 solution.
9. The method for rapid detection of a feline canine pathogen according to claim 8, wherein: the pathogen sample to be detected in the step 1 is body fluid, tissue fluid, secretion fluid of a cat and dog individual to be detected, and is a nasopharyngeal swab, an oral swab or an anal swab.
10. The method for rapid detection of feline-canine pathogens according to claim 9, wherein the primer set specific for each pathogen to be detected in step 3.1 is specifically as follows:
the pathogen to be detected is cat parvovirus, and the specific primer group nucleic acid sequence is shown as SEQ ID NO 1-SEQ ID NO 4, specifically:
upstream outer primer: TGTTTAAACTTGGGCGGG
Downstream outer primer: ATAGTTGTTCCAGCGAACAT
Upstream inner primer: GACAAGAAGCGAATGTTTGTGGGCGTGGTTAAAG
Downstream inner primer: AGAGTGAACCTCTCTTTCCTCAGTATACTGGTT
The pathogen to be detected is canine parvovirus, and the specific primer group nucleic acid sequence is shown in SEQ ID NO 5-SEQ ID NO 8, specifically:
upstream outer primer: AAACTAAAAAGGAAGTGTCA
Downstream outer primer: GTATTAATTCAAATGCT
Upstream inner primer: AACTATCTGGTTGTTGCGGGACTTGGTTAGTAAAAG
Downstream inner primer: GATGGCACAACCAGGAAAAGTCAAAGTACAAATTTC
The pathogen to be detected is feline herpesvirus, the specific primer group nucleic acid sequence is shown in SEQ ID NO 9-SEQ ID NO 12, and specifically:
upstream outer primer: ATGTCTTTTTGAAGTCT
Downstream outer primer: TCACCCATTCACTATCTT
Upstream inner primer: CAGCCTTGATCTTTTCCAGAACCAAATGAATGT
Downstream inner primer: ATGTTTTACTTCATATTTGTCTGCGTAGCC.
11. The method for rapid detection of feline canine pathogens according to claim 10, wherein the pathogen nucleic acid extracted in step 2 comprises DNA and RNA; wherein the DNA comprises genomic DNA, episomal DNA and pathogen DNA of the detected individual, and the RNA comprises total RNA, episomal RNA and pathogen RNA of the detected individual.
12. The method for rapid detection of feline canine pathogens according to claim 11 wherein in step 3.1 the pH of the amplification system is between 2 and 13.
13. A primer composition for rapid detection of a feline canine pathogen for use in the rapid detection method of a feline canine pathogen of claim 1, wherein: comprises at least one group of a feline parvovirus primer group, a canine parvovirus primer group and a feline herpesvirus primer group;
the nucleic acid sequences of the cat parvovirus primer group are shown in SEQ ID NO 1-SEQ ID NO 4, and are respectively:
upstream outer primer: TGTTTAAACTTGGGCGGG
Downstream outer primer: ATAGTTGTTCCAGCGAACAT
Upstream inner primer: GACAAGAAGCGAATGTTTGTGGGCGTGGTTAAAG
Downstream inner primer: AGAGTGAACCTCTCTTTCCTCAGTATACTGGTT
The nucleic acid sequences of the canine parvovirus primer group are shown in SEQ ID NO 5-SEQ ID NO 8, and are respectively:
upstream outer primer: AAACTAAAAAGGAAGTGTCA
Downstream outer primer: GTATTAATTCAAATGCT
Upstream inner primer: AACTATCTGGTTGTTGCGGGACTTGGTTAGTAAAAG
Downstream inner primer: GATGGCACAACCAGGAAAAGTCAAAGTACAAATTTC
The nucleic acid sequences of the feline herpesvirus primer group are shown in SEQ ID NO 9-SEQ ID NO 12, respectively:
upstream outer primer: ATGTCTTTTTGAAGTCT
Downstream outer primer: TCACCCATTCACTATCTT
Upstream inner primer: CAGCCTTGATCTTTTCCAGAACCAAATGAATGT
Downstream inner primer: ATGTTTTACTTCATATTTGTCTGCGTAGCC.
14. A kit for rapid detection of a feline-canine pathogen, characterized in that: a primer composition comprising the primer set of claim 15 for rapid detection of a feline canine pathogen.
15. The kit for rapid detection of a feline canine pathogen according to claim 14, wherein: also included is an amplification system comprising 1mM-80mM Tris-HCl, 10mM-500mM KCl, 0.2mM-30mM MgSO4, 10mM-500mM MgCl2, tween-20 at a concentration of 0.001% -20% by volume, 0.5mM-120mM (NH) 4 ) 2 SO 4 0.1M-15M betaine, 0.2mM-5mM dNTP, 1-40U DNA polymerase, 0.1-20U UDG enzyme, 0.01pM-50M pH indicator and DNA polymerase enhancer.
16. The kit for rapid detection of a feline canine pathogen according to claim 15, wherein: the DNA polymerase enhancer is one or two of gelatin and trimethylamine oxide with the mass volume ratio of 0.1-10 percent and 15-100 mM.
17. The kit for rapid detection of a feline canine pathogen according to claim 16, wherein: the dNTPs are dATP, dTTP, dCTP, dGTP and dUTP.
18. The kit for rapid detection of a feline canine pathogen according to claim 17, wherein: the UDG enzyme is uracil-DNA glycosylase.
19. The kit for rapid detection of a feline canine pathogen according to claim 18, wherein: the pH indicator is bromocresol purple, naphtholphthalein, thymol blue, m-cresol purple, phenol red, cresol red or xylenol orange, and combinations thereof.
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