CN117604169B - Primer and kit for detecting respiratory pathogens of cats and preparation method of primer and kit - Google Patents
Primer and kit for detecting respiratory pathogens of cats and preparation method of primer and kit Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/35—Mycoplasma
Abstract
The invention relates to a primer and a kit for detecting a cat respiratory pathogen and a preparation method thereof, belonging to the technical field of biology. The primer for visually and rapidly detecting the catwalk pathogens and the kit containing the primer can be used for efficiently, rapidly and simply detecting common pathogens such as catchment viruses, catchfly bacilli, catchfly chlamydia, catchment mycoplasma and the like under the constant temperature condition. The problems of long consumption and high cost of pathogen extraction in samples in the prior art are solved, and the method has the advantages of rapidness, high efficiency and reliability, has higher practical value and market competitiveness, can be widely applied to detection and research of cat respiratory pathogens, and has larger market prospect and economic value.
Description
Technical Field
The invention relates to a primer and a kit for detecting a cat respiratory pathogen and a preparation method thereof, belonging to the technical field of biology.
Background
Cats are ideal indoor pets because they are relatively small and do not require a large amount of space. This makes cats an ideal choice for urban residents, especially those residing in apartments or confined spaces. In particular, periodic walking every day is not required, and thus cats are ideal partners for those with independent or busy lifestyles. Cats are more susceptible to respiratory pathogens because of their lower immunity due to the single living conditions and less exercise. There are some major pathogens in the respiratory tract of cats, including feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia and feline mycoplasma, which pose a certain hazard to the health of cats.
Feline calicivirus (Feline Calicivirus, FCV) is a common feline respiratory virus that can cause upper respiratory infections and oral inflammation in cats. Cats infected with feline calicivirus can exhibit sneezing, tearing, loss of appetite, and the like. In severe cases, cats may develop complications such as canker sores and arthritis. Monitoring for the presence of feline calicivirus is important for timely precautionary action, diagnosis and treatment of infected cats. The feline herpesvirus type I (Feline Herpesvirus Type, FHV-1) is also called feline rhinovirus, and cats infected with the feline herpesvirus can have sneeze, runny nose, ocular inflammation and other symptoms. In severe cases, cats may develop keratitis, conjunctivitis and respiratory complications. Bordetella (Bordetella felis) is a bacterium that, often along with other respiratory pathogens, infects the respiratory tract of cats. Chlamydia felis (Chlamydophila felis) and Mycoplasma felis (Mycoplasma felis) are common pathogens that cause upper respiratory tract infections in cats, and the infected cats may develop symptoms such as coughing, sneezing, runny nose, etc.
Timely discovery of infection can help veterinarians take appropriate therapeutic measures, alleviate symptoms of cats and promote recovery. Monitoring for the presence of pathogens can provide important epidemiological data that helps to understand the pathogen's transmission pathways and impact ranges, thereby developing preventive and control measures. In addition, for cat breeders, knowledge of the health of the cat can help them better care for their own pets, reducing the risk of disease transmission. Therefore, monitoring for the presence of the five pathogens above is of great importance to the health of cats and the management of pet cat populations. At present, the detection method of the cat respiratory tract pathogen mainly comprises traditional PCR and real-time fluorescence PCR. However, these methods are cumbersome to operate and are not conducive to rapid and convenient detection.
The LAMP (Loop-mediated isothermal amplification) amplification technology is a nucleic acid amplification technology carried out under the constant temperature condition, double-stranded DNA is in the dynamic balance of unwinding and combining at the temperature of 60-65 ℃, bst polymerase has both strand displacement activity and polymerase activity, and a template can be rapidly, efficiently and specifically amplified under the constant temperature condition without PCR thermal denaturation amplification cycle. The advantages of LAMP over PCR technology are represented by the following: first, LAMP amplification is performed under isothermal conditions, without requiring complicated temperature cycling equipment. In contrast, PCR reactions require precise control of the variation of different temperatures, and require much time and money to ensure the accuracy of the reaction temperature. Only one thermostat is needed for LAMP amplification to complete the amplification reaction, thus being simpler and more economical. Secondly, LAMP amplification has higher amplification efficiency and speed. Because 4-6 primers and Bst DNA polymerase are adopted in the LAMP reaction, a large amount of amplification products can be generated in a short time.
Experimental results indicate that LAMP amplification is faster, and that obvious amplification products can be obtained in 30 minutes to 1 hour, whereas PCR reactions typically take 1-2 hours. LAMP amplification has higher specificity to a target sequence, and LAMP reaction uses a plurality of primers, so that different areas of a plurality of target sequences can be identified simultaneously, thereby improving the specificity. In contrast, PCR reactions typically use only two primers, and may suffer from non-specific amplification problems. In addition, LAMP amplification has the advantage of direct visualization, and a pH indicator is usually added in LAMP amplification reaction, so that a large amount of H is generated in the reaction + The pH of the reaction system is reduced, so that the color-changing amplification can be carried out under the condition of matching with a pH sensitive buffer solution, and the LAMP amplification result can be directly observed through naked eyes without using expensive instruments for detection. Finally, LAMP amplification is highly tolerant to analysis of complex samples. Since the LAMP reaction uses a plurality of primers, the effect of the inhibitor substance present in the sample on the amplification can be offset. This makes the application of LAMP amplification in complex biological samples (e.g., blood, urine, food, etc.) more reliable and stable.
In view of the recent reports on the market, isothermal amplification kits for simultaneously detecting feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia and feline mycoplasma are currently available. Therefore, aiming at pathogens such as feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia and feline mycoplasma in the catwalk, it is necessary to provide a primer and a kit product for detection, so as to solve the problems of inconvenient real-time monitoring, high detection cost and lack of combined detection of the feline respiratory pathogens in the market in the prior art, solve the problem of rapid detection of the feline respiratory pathogens in the prior art, shorten the extraction time consumption and reduce the detection cost.
Disclosure of Invention
In order to solve the problems, the primer for detecting the pathogens of the respiratory tract of the cat and the kit containing the primer are provided, so that common pathogens such as the feline calicivirus, the feline herpesvirus, the feline bordetella, the feline chlamydia and the feline mycoplasma can be efficiently, quickly and simply detected under the constant temperature condition, the problems of long consumed time and high cost for extracting the pathogens in a sample in the prior art are solved, and the primer has the advantages of rapidness, high efficiency and reliability, and has higher practical value and market competitiveness.
According to one aspect of the application, there is provided a primer for detecting a respiratory pathogen of a cat, the primer comprising one or more of the following 1) to 5) sets, each set comprising an inner primer FIP/BIP, an outer primer F3/B3 and a loop primer LF/LB, respectively;
1) Feline calicivirus group: FIP is shown as SEQ ID No.1, BIP is shown as SEQ ID No.2, F3 is shown as SEQ ID No.3, B3 is shown as SEQ ID No.4, LF is shown as SEQ ID No.5, LB is shown as SEQ ID No. 6;
2) Feline herpesvirus group: FIP is shown as SEQ ID No.7, BIP is shown as SEQ ID No.8, F3 is shown as SEQ ID No.9, B3 is shown as SEQ ID No.10, LB is shown as SEQ ID No. 11;
3) Group of bordetella: FIP is shown as SEQ ID No.12, BIP is shown as SEQ ID No.13, F3 is shown as SEQ ID No.14, B3 is shown as SEQ ID No.15, LF is shown as SEQ ID No.16, LB is shown as SEQ ID No. 17;
4) Group of cat chlamydia: FIP is shown as SEQ ID No.18, BIP is shown as SEQ ID No.19, F3 is shown as SEQ ID No.20, B3 is shown as SEQ ID No.21, LB is shown as SEQ ID No. 22;
5) Mycoplasma cat group: FIP is shown as SEQ ID No.23, BIP is shown as SEQ ID No.24, F3 is shown as SEQ ID No.25, B3 is shown as SEQ ID No.26, and LB is shown as SEQ ID No. 27.
Wherein the feline herpesvirus group, the feline chlamydia group, and the feline mycoplasma group do not contain an LF primer sequence.
According to another aspect of the present application, there is provided a kit for detecting a feline respiratory pathogen, characterized in that the kit comprises Bst enzyme, dNTP, magnesium ions and the primer for detecting a feline respiratory pathogen of claim 1.
Optionally, the Bst enzyme dosage used in the kit in combination with each group of primers is 4-8U, and the Bst enzyme dosage is calculated according to a 50 mu l system.
Optionally, the Bst enzyme is used in an amount selected from the group consisting of 4U, 5U, 6U, 7U, 8U and any value therebetween.
Optionally, the concentration of dNTPs matched with each group of primers in the kit is 1-3.0 mM.
Optionally, the dNTP concentration is selected from 1.0 mM, 1.25 mM, 1.5 mM, 1.75 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3 mM, and any value therebetween.
Optionally, the concentration of magnesium ions matched with each group of primers in the kit is 4-8 mM.
Optionally, the selected magnesium ion concentration is selected from the group consisting of 4 mM, 5 mM, 6 mM, 7 mM, 8 mM and any value therebetween.
Optionally, the reagent matched with each group of primers in the kit further comprises phenol red, and the concentration of the phenol red in each group is 20-80 mu M.
By adding phenol red into the amplification system, the amplification result can be visually displayed, so that the detection result can be conveniently judged by naked eyes to compare colors. Because phenol red is slightly soluble in water, when phenol red reagent is prepared, the phenol red reagent reaches a saturated state after exceeding 500 mu M, and the concentration of phenol red added into an amplification system is set to be lower than the range of 500 mu M.
Optionally, the phenol red concentration is selected from 20 [ mu ] M, 25 [ mu ] M, 30 [ mu ] M, 35 [ mu ] M, 40 [ mu ] M, 45 [ mu ] M, 50 [ mu ] M, 55 [ mu ] M, 60 [ mu ] M, 65 [ mu ] M, 70 [ mu ] M, 75 [ mu ] M, 80 [ mu ] M and any value therebetween.
Preferably, the phenol red concentration is 50 mu M.
Optionally, the kit further comprises a sample release agent comprising: 1-5M guanidine hydrochloride, 10-20 mM Tris, 0.5-1 g/L adsorbent resin, 0.01-0.5% polysorbate 20.
Optionally, the guanidine hydrochloride concentration is selected from 1M, 2M, 3M, 4M, 5M and any value therebetween; the Tris concentration is selected from 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM and any value therebetween; the concentration of the adsorption resin is selected from 0.5 g/L, 1.0 g/L, 1.5 g/L, 2.0 g/L, 2.5 g/L and 3.0 g/L and any value therebetween; the polysorbate 20 mass concentration is selected from 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and any value therebetween.
Preferably, the concentration of the adsorption resin is 1 g/L.
Preferably, the polysorbate 20 concentration is 0.1%.
Optionally, the adsorption resin is XAD-7.
According to yet another aspect of the present application, there is provided a kit for the visual detection of a respiratory pathogen in a cat, the kit comprising a feline calicivirus detection tube, a feline herpesvirus detection tube, a feline bordetella detection tube, a feline chlamydia detection tube, a feline mycoplasma detection tube, and a negative colorimetric tube;
the feline calicivirus test tube comprising the feline calicivirus set primer of claim 1, the feline herpesvirus test tube comprising the feline herpesvirus set primer of claim 1, the feline bordetella test tube comprising the feline bordetella set primer of claim 1, the feline chlamydia test tube comprising the feline chlamydia set primer of claim 1, the feline mycoplasma test tube comprising the feline mycoplasma set primer of claim 1;
each detection tube also comprises 50 μl of LAMP reaction solution, wherein the LAMP reaction solution comprises: bst polymerase 4-8U, betaine 0.5-2 mu. L, dNTP mixed solution 1-3.0 mM, magnesium ion 4-8 mM and phenol red 20-80 mu M;
the kit further comprises a sample release agent comprising: 1-5M guanidine hydrochloride, 10-20 mM Tris, 0.5-3 g/L adsorption resin, 0.01-0.5% polysorbate 20;
the kit also comprises liquid paraffin.
Preferably, the phenol red concentration is 50 mu M.
Preferably, the concentration of the adsorption resin is 1 g/L.
Preferably, the polysorbate 20 concentration is 0.1%.
According to a further aspect of the present application, there is provided a method of preparing any one of the above kits.
According to still another aspect of the present application, there is provided a preparation method of the above kit, wherein the LAMP reaction solution further includes a lyoprotectant, and the preparation method includes a step of preparing the LAMP reaction solution into a lyophilized pellet.
According to a further aspect of the present application there is provided the use of the above-described introduction in the manufacture of a product for detecting a respiratory pathogen in a cat.
Benefits of the present application include, but are not limited to:
1. according to the kit for detecting the pathogens of the cat respiratory tract, 5 common pathogens of the cat respiratory tract, namely, the feline calicivirus, the feline herpesvirus, the feline bordetella, the feline chlamydia and the feline mycoplasma, are detected efficiently, quickly and conveniently under the constant temperature condition.
2. According to the kit for detecting the respiratory pathogens of the cats, the detection rate of target sequences in samples is greatly increased by matching with the direct-amplification nucleic acid releasing agent, the detection sensitivity is improved, and the detection sensitivity reaches 10 2 The copies/[ mu ] L; in addition, by utilizing the design of 4-6 specific primers, nonspecific amplification of other related viruses, bacteria and fungi can be avoided, and the amplification of feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia and feline count can be realizedAnd the high specificity detection of the original body is realized, and the detection result is accurate and reliable.
3. According to the kit for detecting the cat respiratory tract pathogen, provided by the invention, the kit can be compatible with a real-time fluorescent instrument, a PCR instrument and a FireMini-8 isothermal nucleic acid amplification instrument for amplification detection, and is matched with liquid paraffin for sealing during amplification, so that aerosol pollution is greatly reduced, detection can be performed in a conventional laboratory or household, high operability and universality are achieved, and a more convenient, rapid, simple and reliable method is provided for cat respiratory tract pathogen detection.
4. According to the kit for detecting the respiratory pathogens of cats, phenol red with a pH indicating effect is added into the reagent, and a large amount of H is generated in the reaction + When the pH of the reaction system is reduced, the reaction liquid is red when the reaction system is not amplified, and if the target sequence exists in the reaction after the reaction is finished, the reaction liquid is yellow, so that the LAMP amplification result can be directly observed by naked eyes without using an expensive instrument for detection.
5. According to the kit for detecting the cat respiratory tract pathogen, provided by the invention, the problems of inconvenient real-time monitoring, high detection cost and lack of cat respiratory tract pathogen combined detection products in the market in the prior art are solved, and the kit can be widely applied to detection and research of the cat respiratory tract pathogen and has a large market prospect and economic value.
6. According to the kit for detecting the respiratory pathogens of the cats, the direct amplification method is used for extracting and releasing nucleic acid from the sample, so that the kit has the advantages of being rapid, efficient, reliable and the like, solves the problems of long consumed time and high cost in pathogen extraction in the sample, and has high practical value and market competitiveness.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 is a diagram of the results of screening for different primer combinations according to the examples of the present application;
FIG. 2 is a diagram showing the results of primer specificity verification according to the examples of the present application;
FIG. 3 is a graph of amplification results of samples extracted using a direct amplification nucleic acid releasing agent and a kit in optimizing the direct amplification nucleic acid releasing agent according to an embodiment of the present application;
FIG. 4 is a schematic diagram of an arrangement for rapid detection of 5 cat respiratory pathogens according to an embodiment of the present application;
FIG. 5 is a graph of the detection results of the sensitivity of the visual rapid detection kit for 5 cat respiratory pathogens according to the embodiment of the present application;
FIG. 6 is a graph I of the detection results of a real sample of a visual rapid detection kit for 5 cat respiratory pathogens according to the embodiment of the present application;
fig. 7 is a second graph of the detection results of a real sample of the visual rapid detection kit for 5 cat respiratory pathogens according to the embodiment of the present application.
Detailed Description
The present application is described in detail below with reference to examples, but the present application is not limited to these examples, and the raw materials and catalysts in the examples of the present application are commercially available unless otherwise specified.
Aiming at 5 respiratory pathogens of combination of feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia and feline mycoplasma, 5 sets of nucleic acid sequences for specifically detecting pathogens are designed. Wherein FIP/BIP is an inner primer, F3/B3 is an outer primer, and LF/LB is a loop primer.
The invention also provides a kit for detecting 5 cat respiratory pathogens. The kit comprises freeze-dried balls (primers, enzymes, buffers and other reagents) required by the LAMP reaction system, and a nucleic acid releasing agent for nucleic acid extraction and liquid paraffin for sealing. The specific operation is as follows:
s1: swabs under the eyelid, thoroughly rubbed to collect an eye swab sample; the same swab was used to moderately swab oral and nasal secretions and a nasopharyngeal swab sample was collected. After the swab sample is collected, the swab head should be broken off in the nucleic acid releasing agent rapidly, shake for 10 seconds and mix thoroughly, and left at room temperature for 5 minutes, so that the pathogen on the swab head is dissolved into the nucleic acid releasing agent thoroughly.
S2: the reagent preparation is carried out according to the following steps: 1 part of amplified freeze-dried ball is contained in an eight-connecting tube, and 50 mu L/hole of a detection sample and 50 mu L/hole of liquid paraffin are contained;
s3: placing the eight-connecting tube into a FireMini-8 isothermal nucleic acid amplification instrument to perform nucleic acid amplification reaction; after the reaction is finished and the temperature is reduced to room temperature, the reaction is placed in an environment with good light for observation.
Interpretation of the results: the reaction liquid is red and negative, and the reaction liquid is yellow and positive.
The following describes the embodiments of the present invention by way of specific examples.
Example 1 design and screening of primers
Specific primers were designed on PrimerExplorer V5 (http:// Primerexplorer. Jp/e /) website based on the genes of each pathogen in GenBank and screened according to the principle of specific primer sequence construction. Standards for each pathogen are plasmids synthesized by biological companies. A plurality of pairs of primers are designed for each pathogen to screen, primers with good specificity, no cross reaction and good amplification efficiency are selected, and the test primers are shown in Table 1.
TABLE 1 list of test primers to be selected
TABLE 1 Table 1 to Table 1
TABLE 1 followed by TABLE 2
The binding efficiency of different primers and templates is different, and the final color change is greatly different in the visualized LAMP reaction. For example, the same concentration of template, three different primers, will appear in different degrees of color (yellow, orange, red), the more yellow the color indicates that this primer is easier to bind to the template, the better the design is, and vice versa, the worse the result is seen in FIG. 1.
In FIG. 1, the primers for detecting feline calicivirus are taken as examples, and it can be seen from the amplification results, the amplification time and the color that the amplification efficiency is inferior to that of the No.4 FCV primer and it is difficult to obtain a high-sensitivity detection result, although the negative-positive contrast of the amplification results is obvious after the No.1 FCV and No.3 FCV primers are used. And after adopting No.2 FCV, no.5 FCV and No.6 FCV primers, the constant temperature amplification is carried out for 30 minutes at 65 ℃, and the contrast of yin and yang is not obvious, so that the No.4 FCV primer is screened out as an amplification primer combination of the kit. The detection of the herpes cat virus, the bordetella cat, the chlamydia cat and the mycoplasma cat shows the same result, and the primer of the invention does not generate cross reaction, so the sensitivity is higher and the specificity is better. However, the other primer combinations in FIG. 1 are poor in amplification sensitivity and specificity, and detection of the respiratory pathogens of cats cannot be achieved. Therefore, the five combinations of No.4 FCV, no.6 FHV, no.2 cat wave, no.1 CP and No.2 MP are selected for specificity and sensitivity detection in the subsequent experiments, and the primer information is shown in Table 2.
Table 2 primers selected
Example 2 primer specificity verification
10 selected from the group consisting of feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia, and feline mycoplasma plasmid 5 The copies/. Mu.l served as amplification templates, 2 per template in parallel, giving the amplification results FIG. 2.
As can be seen from the amplification results of fig. 2, after the amplification of different types of plasmid templates by using the primers of the cat respiratory tract pathogens, only the sites (primers) corresponding to the primers and the templates were successfully amplified, and the results showed that the results were yellow positive; other plasmid templates, results were red negative; the specificity of the amplification system is good.
Example 3 optimization of the reaction System
50. Mu.l of LAMP reaction solution was prepared as follows: bst polymerase 4~8U, betaine 1 [ mu ] L, dNTP mixed solution 1~3.0 mM, magnesium ion 4~8 mM, phenol red 50 [ mu ] M, aseptic water fill to 50 [ mu ] L, FIP 1.6.6 [ mu ] M, BIP 1.6.6 [ mu ] M, F3.2 [ mu ] M, B3.2 [ mu ] M, LF 0.4 [ mu ] M, LB 0.4 [ mu ] M.
Since double-stranded DNA is in a stable state of dynamic equilibrium at 65℃and LAMP primers can bind to complementary sequences in a double-stranded target gene, and then DNA synthesis is started by using the strand displacement activity of Bst polymerase, thereby replacing and releasing another single-stranded DNA, the amount of Bst polymerase is very important in amplification. dNTPs are provided as a raw material in an amplification step, and low dNTP concentrations are used in a reaction to reduce the misincorporation of nucleotides during starting and extension at non-target positions, but too low concentration of dNTPs can result in incomplete amplification due to insufficient raw material in the amplification process, so the inventor has searched for the optimal dNTP concentration. Magnesium ion mainly affects the activity of enzyme, and generally, the higher the concentration of magnesium ion is, the higher the amplification efficiency is, but the specificity is reduced; the inventors screened the amount of Bst polymerase, dNTP concentration, magnesium ion concentration optimal for detection of the 5 pathogens of the present invention.
Firstly, optimizing the dosage of Bst polymerase, setting the dosage of Bst polymerase to be 4U, 5U, 6U, 7U and 8U, the amplification temperature to be 65 ℃, the final concentration of dNTP to be 2.5 mM, the final concentration of magnesium ion to be 6 mM, setting the dosage of Bst polymerase to be unchanged except that the dosage of Bst polymerase is changed, setting the amplification instrument to be a FireMini-8 constant temperature nucleic acid amplification instrument, and selecting 10 of feline calicivirus 5 The copies/. Mu.l was used as an amplification template to screen the optimal Bst polymerase amount for amplification to a positive time in 30 minutes and no false positives. Through optimizing the usage amount of Bst polymerase, when other amplification conditions are consistent, and the usage amount of Bst polymerase is 8U, the color change time of the same detection sample in a FireMini-8 isothermal nucleic acid amplification instrument for the occurrence of negative-positive conversion is shortest, the time is 18 minutes, and the color change time of the same detection sample for the occurrence of negative-positive conversion is more than 20 in 4U, 5U, 6U and 7UAnd (3) minutes.
Secondly, optimizing dNTP concentration, setting the dosage of dNTP concentration to be 1.0 mM, 1.25 mM, 1.5 mM, 1.75 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM and 3 mM, the amplification temperature to be 65 ℃, the dosage of Bst polymerase to be 8U, the final concentration of magnesium ion to be 6 mM, setting the other settings to be unchanged except that the dosage of dNTP is changed, and the amplification instrument to be a FireMini-8 constant temperature nucleic acid amplification instrument, and selecting 10 of feline calicivirus 5 The copies/. Mu.l was used as an amplification template to screen for optimal dNTP concentrations at a rate of amplification positive time in 30 minutes without false positives. Through optimizing the concentration of dNTPs, when other amplification conditions are consistent, the concentration of dNTPs is 2.5 mM, 2.75 mM and 3 mM, the color change time of the same detection sample in a FireMini-8 isothermal nucleic acid amplification instrument for negative-positive conversion is shortest, the time is 18 minutes, and the concentration of dNTPs is 1.0 mM, 1.25 mM and 1.5 mM, and the color change of negative-positive conversion is not generated, and the time is 1.75 mM, 2.0 mM and 2.25 mM and is more than 20 minutes. Based on the principle of saving reagents, the maximum amplification effect is achieved when the final concentration of dNTPs is 2.5 and mM.
Optimizing magnesium ion, setting magnesium ion concentration to be 4 mM, 5 mM, 6 mM, 7 mM and 8 mM, amplifying temperature to be 65 ℃, bst polymerase dosage to be 8U, dNTP final concentration to be 2.5 mM, setting magnesium ion dosage to be unchanged, setting the rest to be unchanged, amplifying instrument to be FireMini-8 isothermal nucleic acid amplifier, selecting 10 of cat calicivirus 5 The copies/. Mu.l was used as an amplification template to screen for optimal magnesium ion concentration at a rate of amplification positive time in 30 minutes without false positives. Through optimizing the magnesium ion concentration, when other amplification conditions are consistent, the magnesium ion concentration is 6 mM, the color change time of the same detection sample for negative-positive conversion in a FireMini-8 isothermal nucleic acid amplification instrument is shortest, the time is 18 minutes, the color change of negative-positive conversion does not occur in the magnesium ion concentration of 4 mM, the positive time of the magnesium ion concentration of 5 mM is more than 20 minutes, and the false positive occurs after the amplification of the magnesium ion concentration of 7 mM and 8 mM is finished for 30 minutes. The final optimized reaction system results are shown in Table 3.
TABLE 3 50. Mu.l reaction system composition
Wherein the buffer Mix contains 1% Tween-20, 100 mM (NH 4 ） 2 SO 4 500 mM KCl,200 mM Tris-HCl; the 10 XPrimer contained 16. Mu.M FIP/BIP, 2. Mu. M F3/B3, 4. Mu.M LF/LB.
Example 4 optimization of the Release agent for direct nucleic acid
The direct amplification nucleic acid releasing agent is used for directly cracking nucleic acid from a sample without extracting the nucleic acid, and the components of the nucleic acid releasing agent are used for ensuring the effective release of the nucleic acid in the sample and cannot influence the amplification of subsequent LAMP. The nucleic acid releasing agent can omit the step of extracting nucleic acid, so as to simplify the experimental operation flow, save the experimental time and accelerate the experimental process. The conventional nucleic acid extraction process includes a plurality of steps such as cell lysis, nucleic acid precipitation, washing, etc., and the nucleic acid releasing agent is capable of directly cleaving nucleic acid from a sample without such troublesome steps, thereby saving time required for the experiment.
In order to achieve the above object, the present invention provides a nucleic acid releasing agent which can rapidly release nucleic acids of pathogens in a swab and remove most of amplification inhibitors, and can be used for direct amplification to analyze nucleic acids of a cat respiratory swab sample.
Since the present amplification reagents are based on a change in pH, the pH of the sample releasing agent is 8.5.+ -. 0.5 (slightly alkaline). The sample release agent comprises: 1-5. 5M guanidine hydrochloride, 10-20 mM Tris, adsorption resin XAD-7 (1 g/L), 0.1% polysorbate 20 and an appropriate amount of water. The system is added with a proper amount of guanidine hydrochloride and polysorbate 20, so that the pathogen cells can be fully lysed, and the condition of missed detection of a sample can be avoided; the addition of a proper amount of Tris is used for ensuring that the nucleic acid of the pathogen is not degraded in a relatively stable environment within a short time; the adsorption resin XAD-7 is added to adsorb and settle impurities such as mucin in the sample, so that the amplification reaction can be smoothly carried out without being influenced by other impurities; 10 of feline calicivirus 5 copies/. Mu.l plasmid and healthy cat swab samplesMixing, preparing a solution according to guanidine hydrochloride 1M, tris 10 mM, an adsorption resin XAD-7 (1 g/L) and polysorbate 20 0.1%, preparing according to the amplification system in example 3, detecting a sample in a FireMini-8 isothermal nucleic acid amplification apparatus, wherein the amplification time is about 20 minutes, and the amplification time is equivalent to the result of amplifying only pure plasmids; similarly, the amplification time was about 20 minutes, which is equivalent to the result of amplifying only the pure plasmid, when the solution was prepared from 5M guanidine hydrochloride, 20 mM Tris, adsorbent resin XAD-7 (1 g/L) and 0.1% polysorbate 20.
Based on the above experimental findings, 1M guanidine hydrochloride, 10 mM Tris, adsorption resin XAD-7 (1 g/L), 0.1% polysorbate 20 were solution formulated for nucleic acid direct amplification release agent. Taking a pet cat sample positive for qPCR detection results including cat calicivirus, cat mycoplasma and cat herpesvirus, taking an eye and nose throat swab, placing one sample into 500 mu l of a nucleic acid releasing agent, incubating for 5 minutes at room temperature after shaking to obtain an amplified sample, preparing according to the system of the example 4, adding 5 mu l of the sample, and detecting the sample in a FireMini-8 isothermal nucleic acid amplification instrument; one sample was subjected to nucleic acid extraction using a viral DNA/RNA extraction kit (magnetic bead method) kit of well known medicine, and the extracted sample was prepared as an amplified sample according to the system of example 4, and 5. Mu.l of the sample was added thereto, and the sample was detected in a FireMini-8 isothermal nucleic acid amplification apparatus. After 30 minutes of amplification, the results are shown in FIG. 3.
From the results of fig. 3, it can be concluded that: in the amplification time of 30 minutes, the consistency rate of the sample result amplified by using the direct amplification nucleic acid releasing agent and the detection result of the gold standard qPCR is 100%; the consistency of the detection results of the sample feline calicivirus and feline herpesvirus extracted by using the kit and the gold standard qPCR is 100%, and 1 template is not detected in three parallels of the feline mycoplasma. The nucleic acid releasing agent provided by the application can directly cleave nucleic acid in a sample and release the nucleic acid into a solution, so that the yield of the effectively amplified nucleic acid can be improved. In contrast, there may be some loss during the nucleic acid extraction process, resulting in a relatively low yield of nucleic acid. The direct amplification nucleic acid releasing agent provided by the application only cleaves nucleic acid in a sample, and does not affect the structure and function of the nucleic acid, so that the nucleic acid releasing agent does not affect subsequent LAMP amplification when the sample is cleaved.
Example 5 establishment of a kit for visual rapid detection of 5 cat respiratory pathogens
Lyophilization was performed following the procedure described below in connection with the system in example 3: 50 μl of LAMP freeze-dried reaction solution was prepared, and the ratio was as follows: bst polymerase 8U, betaine 1 [ mu ] L, dNTP mixed liquor 2.5 mM, magnesium ion 6 mM, phenol red 50 [ mu ] M, sterile water is filled up to 50 [ mu ] L, FIP 1.6 [ mu ] M, BIP 1.6.6 [ mu ] M, F3.2 [ mu ] M, B, 0.2 [ mu ] M, LF, 0.4 [ mu ] M, LB, 0.4 [ mu ] M, RT enzyme 75U, and lyoprotectant 20 [ mu ] L, and RT enzyme is added in the lyophilization for reverse transcription of RNA due to the presence of DNA and RNA in a real sample. Preparing a reagent according to the formula, filling the reagent into a bead dropping machine, dropping the bead dropping machine into liquid nitrogen according to the concentration of 50 mu L/drop, rapidly transferring the pre-frozen microspheres into a pre-frozen penicillin bottle, semi-capping the pre-frozen microspheres into a pre-frozen vacuum freeze dryer, performing vacuum freeze drying, and sub-packaging the pre-frozen microspheres into 0.2 ml transparent eight rows according to the arrangement mode shown in figure 4 after obtaining the freeze-dried microspheres.
Example 6 visual sensitivity detection of kit for rapid detection of 5 cat respiratory pathogens
The plasmid obtained in example 1 was isolated from the original plasmid 10 10 After 10-fold gradient dilution of the peptides/MuL, the following steps are carried out: 10 6 copies/µL、10 5 copies/µL、10 4 copies/µL、10 3 copies/µL、10 2 copies/µL、10 1 copies/µL、10 0 The copies/. Mu.L. According to the lyophilized pellet obtained in step 3, 50. Mu.l was added to obtain the following amplification results of FIG. 5. An eight row left to right sequence was consistent with the lyophilization sites of example 5, with the following sequences from left to right: feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia, feline mycoplasma, negative cuvette.
As can be seen from the results of FIG. 5, as a combined detection kit for detecting pathogens of the respiratory tract of cats, feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia, and feline mycoplasma can be stably detected to 10 2 copies/μl. Mycoplasma cat and chlamydia cat freeze-dried ball amplification 10 1 At copies/. Mu.l, the amplification result was red negative.
Example 7 detection of a real sample of a kit for visual rapid detection of 5 cat respiratory pathogens
A swab sample of 30 cats was collected from a cat coffee shop, one swab sample of the same cat containing three parts of eye, nose and mouth, operated as follows:
s1, wiping the eye swab under an eyelid with the swab, and collecting an eye swab sample; the same swab was used to moderately smear the oral and nasal secretions of the cat and a nasopharyngeal swab sample was collected. After the swab sample is collected, the swab head should be broken off in the nucleic acid releasing agent rapidly, shake for 10 seconds and mix thoroughly, and left at room temperature for 5 minutes, so that the pathogen on the swab head is dissolved into the nucleic acid releasing agent thoroughly.
S2, preparing a reagent: one octal row of amplification sites from left to right was feline calicivirus, feline herpesvirus, feline bordetella, feline chlamydia, feline mycoplasma, wherein each octal contained 1 amplified lyophilized pellet and 50 μl/well of test sample and 50 μl/well of liquid paraffin.
S3, placing the eight-connecting tube into a FireMini-8 isothermal nucleic acid amplification instrument to perform nucleic acid amplification reaction; after the reaction is finished and cooled to room temperature, the reaction product is placed in an environment with good light for observation, and the reaction result is shown in fig. 6 and 7.
From the results of FIGS. 6 and 7, 15 out of 30 cats were completely healthy (50%), 11 infected with Mycoplasma (36.7%), 9 infected with Carnitium catwalk (30%), 4 infected with Calcivirus (13.3%), 3 infected with Chlamydia feline (10%), 2 infected with feline herpesvirus (6.7%). Among 15 cats with respiratory diseases detected, 5 cats showed severe sneezing, tearing, and the other 10 had no obvious symptoms. The qPCR recheck is used for 30 cat samples, the coincidence rate of the detection result and the visual kit for rapidly detecting 5 cat respiratory pathogens is 100%, and the accuracy of the kit is proved to be consistent with that of qPCR.
The foregoing is merely exemplary of the present application, and the scope of the present application is not limited to the specific embodiments, but is defined by the claims of the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.
Claims (4)
1. The kit for visually detecting the pathogens of the respiratory tract of the cat is characterized by comprising a cat cup virus detection tube, a cat herpesvirus detection tube, a catwalk bacillus detection tube, a cat chlamydia detection tube, a cat mycoplasma detection tube and a negative colorimetric tube; the feline calicivirus detection tube comprises a feline calicivirus group primer, the feline herpesvirus detection tube comprises a feline herpesvirus group primer, the feline bordetella detection tube comprises a feline chlamydia group primer, the feline chlamydia detection tube comprises a feline mycoplasma group primer, each group of the primers respectively comprises an inner primer FIP/BIP, an outer primer F3/B3 and a loop primer LF/LB, and the specific sequences contained in the primers are shown in the following 1) -5);
1) Feline calicivirus group: FIP is shown as SEQ ID No.1, BIP is shown as SEQ ID No.2, F3 is shown as SEQ ID No.3, B3 is shown as SEQ ID No.4, LF is shown as SEQ ID No.5, LB is shown as SEQ ID No. 6;
2) Feline herpesvirus group: FIP is shown as SEQ ID No.7, BIP is shown as SEQ ID No.8, F3 is shown as SEQ ID No.9, B3 is shown as SEQ ID No.10, LB is shown as SEQ ID No. 11;
3) Group of bordetella: FIP is shown as SEQ ID No.12, BIP is shown as SEQ ID No.13, F3 is shown as SEQ ID No.14, B3 is shown as SEQ ID No.15, LF is shown as SEQ ID No.16, LB is shown as SEQ ID No. 17;
4) Group of cat chlamydia: FIP is shown as SEQ ID No.18, BIP is shown as SEQ ID No.19, F3 is shown as SEQ ID No.20, B3 is shown as SEQ ID No.21, LB is shown as SEQ ID No. 22;
5) Mycoplasma cat group: FIP is shown as SEQ ID No.23, BIP is shown as SEQ ID No.24, F3 is shown as SEQ ID No.25, B3 is shown as SEQ ID No.26, LB is shown as SEQ ID No. 27;
each detection tube further comprises an LAMP reaction liquid, and the LAMP reaction liquid comprises Bst polymerase, betaine, dNTP mixed liquid, magnesium ions, phenol red and RT enzyme according to a 50-mu l system, wherein Bst enzyme is 7-8U, dNTP concentration is 2.5-3.0 mM, magnesium ion concentration is 5-6 mM, and phenol red concentration is 20-80 mu M;
the kit further comprises a sample release agent comprising: 1-5M guanidine hydrochloride, 10-20 mM Tris, 0.5-3 g/L adsorption resin XAD-7, and 0.01-0.5% polysorbate 20.
2. The kit for visually detecting a feline respiratory pathogen according to claim 1, wherein the LAMP reaction solution comprises: bst polymerase 8U, betaine 0.5-2 mu L, dNTP mixed solution 2.5-3.0 mM, magnesium ion 6 mM, phenol red 20-80 mu M, RT enzyme 75U.
3. The kit for the visual detection of a feline respiratory pathogen according to claim 1, wherein the kit further comprises liquid paraffin.
4. The method for preparing the kit according to any one of claims 1 to 3, further comprising a lyoprotectant in the LAMP reaction solution in the method for preparing the kit, wherein the method for preparing the kit comprises the step of preparing the LAMP reaction solution into lyophilized balls.
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| CN111455107A (en) * | 2020-04-13 | 2020-07-28 | 杭州博日科技有限公司 | Primer group, product, method and application for detecting feline respiratory pathogens |
| CN112593014A (en) * | 2020-12-31 | 2021-04-02 | 上海基灵生物科技有限公司 | Nucleic acid composition, kit and method for detecting pathogens of upper respiratory diseases of cats |
| CN116622909A (en) * | 2023-04-26 | 2023-08-22 | 厦门宝太生物科技股份有限公司 | Isothermal amplification detection reagent and detection method for feline herpesvirus I type |
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| CN112593014A (en) * | 2020-12-31 | 2021-04-02 | 上海基灵生物科技有限公司 | Nucleic acid composition, kit and method for detecting pathogens of upper respiratory diseases of cats |
| CN116622909A (en) * | 2023-04-26 | 2023-08-22 | 厦门宝太生物科技股份有限公司 | Isothermal amplification detection reagent and detection method for feline herpesvirus I type |
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