CN111455107A

CN111455107A - Primer group, product, method and application for detecting feline respiratory pathogens

Info

Publication number: CN111455107A
Application number: CN202010287980.3A
Authority: CN
Inventors: 田星; 陈芝娟; 王虹军; 贺贤汉
Original assignee: Hangzhou Bioer Technology Co ltd
Current assignee: Hangzhou Bioer Technology Co ltd
Priority date: 2020-04-13
Filing date: 2020-04-13
Publication date: 2020-07-28

Abstract

The invention provides a primer group for detecting a feline respiratory pathogen, a product, a method and application, and relates to the technical field of nucleic acid detection. The primer group provided by the invention can be used for identifying 5 cat respiratory pathogens of the feline herpesvirus type1, the feline calicivirus, the mycoplasma, the feline chlamydia and the bordetella bronchiseptica in the same reaction system, and compared with the traditional method, the detection cost and time are greatly reduced.

Description

Primer group, product, method and application for detecting feline respiratory pathogens

Technical Field

The invention relates to the technical field of nucleic acid detection, in particular to a primer group, a product, a method and application for detecting a feline respiratory pathogen.

Background

Feline herpesvirus type1 (Feline herpesvirus type1, FHV1) is the etiological agent of Feline viral rhinotracheitis, belongs to the α herpesvirus subfamily and the varicella in the herpesvirus family, and can cause acute and highly-contact upper respiratory diseases of felines.

Feline Calicivirus (FCV), a member of the genus Calicivirus, belongs to the family Feline caliciviridae, is a single-stranded RNA virus that is non-enveloped and has a spherical capsid (with multiple goblet pits on the capsid), is highly contagious, can cause moderate, self-limiting, multiple oral problems and upper respiratory diseases, is one of the most common causative agents of Feline infectious upper respiratory diseases, and can be virulent for life in a diseased cat. The main clinical symptoms are upper respiratory tract symptoms, fever, nasal secretion increase, conjunctivitis, mental depression, salivation, oral ulcer and the like, and are similar to the symptoms of feline infectious rhinotracheitis caused by feline herpesvirus type1 and difficult to distinguish clinically. In addition to this, feline calicivirus infection can lead to kitten lameness syndrome or virulent systemic feline calicivirus, the latter of which is often lethal.

Mycoplasma (Mycoplasma) belongs to a tiny prokaryote without a cell wall, can pass through a bacterial filter, is a microorganism independently living between viruses and bacteria, and is gram-negative. Mycoplasma in cats primarily infect the eyes and upper respiratory tract of cats and can spread between cats, causing severe edema and mild rhinitis in the conjunctiva of cats. According to statistics, mycoplasma can be separated from the throat of 35% of healthy cats, so that mycoplasma can be a opportunistic pathogen and is secondary to inflammation caused by other causes.

Chlamydia felis (Feline Chlamydia) is a gram-negative rod-shaped coccus without a cell wall, is an intracellular parasitic bacterium, mainly parasitizes mucosal tissues and can cause respiratory diseases, but most of the Chlamydia conjunctivitis (also known as Chlamydia conjunctivitis) and conjunctivitis of cats. Since chlamydia felis cannot survive outside the host, infection must be an intimate contact between cats, and thus the condition is common in many cats in intimate contact, and the secretions of the eyes are probably the most important source of infection. Cats can become carriers of asymptomatic infections, but latent infections can be activated to develop clinical symptoms due to stress or immunosuppression.

Bordetella Bronchiseptica (also known as Bordetella Bronchiseptica), a gram-negative aerobic bacterium, can cause occult respiratory infections and acute and chronic inflammation in various mammals, such as pigs, dogs, cats, horses, cattle, rabbits, and mice. Clinical symptoms include sneezing, secretion in the eyes (yellow-green), secretion in the nasal cavity (yellow-green), cough (less common), and less common clinical symptoms include increased lung breathing sound, swollen lymph nodes, fever, pneumonia, dyspnea, etc. Among cats, the incidence of kittens and geriatric cats aged less than 10 weeks is higher.

The pathogens of the above 5 cats belong to respiratory pathogens, the clinical symptoms are similar, and even the pathogens are frequently mixed to cause infection, so that the diagnosis difficulty is increased, and the risk of cat disease aggravation caused by non-symptomatic treatment is increased, so that the detection and identification of the types of the pathogens are very important.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

It is a first object of the present invention to provide a primer set for detecting a feline respiratory pathogen that alleviates at least one of the technical problems of the prior art.

A second object of the invention is to provide a product for detecting a feline respiratory pathogen.

It is a third object of the invention to provide a method for detecting a feline respiratory pathogen.

The fourth purpose of the invention is to provide the application of the primer group, the product or the method in detecting the feline respiratory pathogens.

The invention provides a primer group for detecting a feline respiratory pathogen, which comprises a primer for detecting feline herpes type1 virus, a primer for detecting feline calicivirus, a primer for detecting mycoplasma, a primer for detecting feline chlamydia and a primer for detecting bordetella bronchiseptica;

wherein, the primer for detecting the feline herpes type1 virus has the nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, or the nucleotide sequence which has at least 85 percent of identity with the SEQ ID NO.1 and/or the SEQ ID NO. 2;

the primer for detecting the feline calicivirus has nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4, or nucleotide sequences with at least 85% of identity with SEQ ID NO.3 and/or SEQ ID NO. 4;

the primer for detecting mycoplasma has nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO.6, or nucleotide sequences with at least 85% of identity with SEQ ID NO.5 and/or SEQ ID NO. 6;

the primer for detecting the cat chlamydia has a nucleotide sequence shown as SEQ ID NO.7 and SEQ ID NO.8, or a nucleotide sequence which has at least 85% of identity with the SEQ ID NO.7 and/or the SEQ ID NO. 8;

the primer for detecting the bordetella bronchiseptica has the nucleotide sequences shown as SEQ ID NO.9 and SEQ ID NO.10, or the nucleotide sequences with at least 85% of identity with SEQ ID NO.9 and/or SEQ ID NO. 10.

Further, the primer group also comprises a probe for detecting the feline herpes type1 virus, a probe for detecting the feline calicivirus, a probe for detecting the mycoplasma, a probe for detecting the feline chlamydia and a probe for detecting the bordetella bronchiseptica;

the probe for detecting the feline herpes type1 virus has a nucleotide sequence shown as SEQ ID NO.11, or a nucleotide sequence which has at least 85% of identity with the SEQ ID NO. 11;

the probe for detecting the feline calicivirus has a nucleotide sequence shown as SEQ ID NO.12, or a nucleotide sequence with at least 85% of identity with SEQ ID NO. 12;

the probe for detecting mycoplasma has a nucleotide sequence shown as SEQ ID NO.13, or a nucleotide sequence with at least 85% identity with SEQ ID NO. 13;

the probe for detecting the cat chlamydia has a nucleotide sequence shown as SEQ ID NO.14 or a nucleotide sequence which has at least 85% of identity with the SEQ ID NO. 14;

the probe for detecting bordetella bronchiseptica has a nucleotide sequence shown in SEQ ID No.15, or a nucleotide sequence with at least 85% identity with SEQ ID No. 15.

Furthermore, both ends of the probe for detecting the feline herpes type1 virus, the probe for detecting the feline calicivirus, the probe for detecting the mycoplasma, the probe for detecting the feline chlamydia and the probe for detecting the bordetella bronchiseptica independently contain a fluorescence quenching group and different fluorescence reporter groups respectively;

preferably, the fluorescent reporter group comprises FAM, HEX, ROX, CY5, or CY 5.5; the probe for detecting feline herpes type1 virus preferably comprises FAM, the probe for detecting feline calicivirus preferably comprises HEX, the probe for detecting mycoplasma preferably comprises ROX, the probe for detecting feline chlamydia preferably comprises CY5, and the probe for detecting bordetella bronchiseptica preferably comprises CY 5.5;

preferably, the fluorescence quenching group comprises BHQ1, BHQ2 or BHQ 3.

Further, the working concentration of the primer for detecting the feline herpes type1 virus, the primer for detecting the feline calicivirus, the primer for detecting the mycoplasma, the primer for detecting the feline chlamydia and the primer for detecting the bordetella bronchiseptica is 5-20 nmol;

preferably, the working concentration of the probe for detecting feline herpes type1 virus, the probe for detecting feline calicivirus, the probe for detecting mycoplasma, the probe for detecting feline chlamydia and the probe for detecting bordetella bronchiseptica is 1 to 10 nmol.

The invention also provides a product for detecting the feline respiratory pathogens, which comprises the primer group;

preferably, the product comprises a reagent or kit.

Further, the kit also comprises at least one of a PCR reaction solution, a positive control substance or a negative control substance;

preferably, the positive control comprises a plasmid containing part or all of the feline herpesvirus type1, a plasmid containing part or all of the feline calicivirus, a plasmid containing part or all of the mycoplasma, a plasmid containing part or all of the feline chlamydia, and a plasmid containing part or all of the bordetella bronchiseptica;

preferably, the negative control comprises water, preferably sterile deionized water.

The invention also provides a method for detecting the feline respiratory pathogens, which takes the genome nucleic acid of a sample to be detected as a template, uses the primer group or the product to carry out PCR reaction, and judges whether the sample to be detected contains the feline respiratory pathogens according to the specific segment or the amplification curve contained in the amplification product:

if the amplification product contains a specific fragment or an amplification curve of the feline herpes type1 virus, the sample to be detected contains the feline herpes type1 virus;

if the amplification product contains a specific fragment or an amplification curve of the feline calicivirus, the sample to be detected contains the feline calicivirus;

if the amplification product contains a specific fragment or an amplification curve of the mycoplasma, the sample to be detected contains the mycoplasma;

if the amplification product contains a specific fragment or an amplification curve of the cat chlamydia, the sample to be detected contains the cat chlamydia;

if the amplification product contains a specific fragment or an amplification curve of bordetella bronchiseptica, the sample to be tested contains bordetella bronchiseptica.

Further, the PCR reaction is a fluorescent quantitative PCR reaction;

preferably, the reaction conditions of the fluorescent quantitative PCR include at least one of the following conditions: reverse transcription is carried out for 8-12min at the temperature of 48-52 ℃; pre-denaturation at 92-98 deg.C for 0.5-1.5 min; denaturation at 92-98 deg.C for 8-12 s; annealing at 58-62 deg.C for 15-25 s; a total of 38-42 cycles; setting the temperature to be 58-62 ℃ to collect fluorescence signals of all channels;

preferably, the reaction conditions of the fluorescent quantitative PCR include at least one of the following conditions: reverse transcription is carried out for 10min at 50 ℃; pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 10 s; annealing at 60 ℃ for 20 s; a total of 40 cycles; the fluorescence signal of each channel was collected at 60 ℃.

Further, the Ct value of each channel of the positive control is less than 30, preferably 25 +/-2;

the positive control comprises a plasmid containing part or all of the feline herpes type1 virus, a plasmid containing part or all of the feline calicivirus, a plasmid containing part or all of the mycoplasma, a plasmid containing part or all of the feline chlamydia, and a plasmid containing part or all of the bordetella bronchiseptica.

In addition, the invention also provides application of the primer group, the product or the method in detecting the feline respiratory pathogens.

In the primer group for detecting the feline respiratory pathogens, the primers for detecting the feline herpes type1 virus, the primers for detecting the feline calicivirus, the primers for detecting the mycoplasma, the primers for detecting the feline chlamydia and the primers for detecting the bordetella bronchiseptica are primers designed through highly conserved regions of various pathogens, do not have cross reaction with pathogens of other cats, and have the characteristics of strong specificity and high sensitivity. The primer group provided by the invention can be used for identifying 5 cat respiratory pathogens of herpesvirus catarrhalis type1, feline calicivirus, mycoplasma, feline chlamydia and bordetella bronchiseptica in the same reaction system. When 5 primers are placed in the same reaction system, different primer pairs do not interfere with each other, the result analysis of PCR amplification is not influenced, and the detection cost and time are greatly reduced compared with the traditional method.

According to the method for detecting the feline respiratory pathogens, the primer group or the product provided by the invention is used for carrying out PCR detection on a sample to be detected, and the accurate molecular identification of the feline herpesvirus type1, feline calicivirus, mycoplasma, feline chlamydia and bordetella bronchiseptica is realized according to different detection results.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the optimized result of the fluorescence PCR detection primer probe for detecting herpesvirus catarrhalis type1, feline calicivirus, mycoplasma, feline chlamydia, and Bordetella bronchiseptica provided in the embodiments of the present invention;

FIG. 2 is a diagram showing the results of specificity verification using the primer set provided by the present invention.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

According to one aspect of the invention, a primer set for detecting feline respiratory pathogens is provided, comprising a primer for detecting feline herpes type1 virus, a primer for detecting feline calicivirus, a primer for detecting mycoplasma, a primer for detecting feline chlamydia, and a primer for detecting bordetella bronchiseptica;

In the present invention, "identity" refers to similarity between nucleotide sequences, including nucleotide sequences having at least 85% (for example, but not limited to, 85%, 88%, 90%, 92%, 95%, 98%, 99% or more) identity to the nucleotide sequences represented by SEQ ID NO.1 to 10 described in the present invention, and having the same function. When the nucleotide sequence shown in SEQ ID NO.1-10 is selected as the primer in the primer group provided by the invention, the primer group has stronger specificity and higher sensitivity.

"nucleotide sequence having at least 85% identity with SEQ ID No.1 and/or SEQ ID No. 2" means, for example, a primer for detecting feline herpes type1 virus, which may be SEQ ID No.1 and SEQ ID No.2, or a nucleotide sequence having at least 85% identity with SEQ ID No.2 and SEQ ID No.1, or a nucleotide sequence having at least 85% identity with SEQ ID No.1 and a nucleotide sequence having at least 85% identity with SEQ ID No. 2. The same holds true for the primers used for detecting feline calicivirus, for detecting mycoplasma, for detecting feline chlamydia and for detecting bordetella bronchiseptica.

It should be noted that the primer set provided by the present invention can be used for different types of PCR reactions. Typically, when used in a conventional PCR assay, the presence or absence of a band of an amplification product and the size of a fragment of the amplification product can be used to determine whether a test sample contains herpesvirus felis 1, feline calicivirus, mycoplasma, chlamydia felis, or bordetella bronchiseptica, or to identify which pathogen it contains. Typically, when the PCR amplification product is used for fluorescence quantitative PCR detection, whether a sample to be detected contains the herpesvirus felis 1, the calicivirus felis, the mycoplasma, the chlamydia felis or the bordetella bronchiseptica or which pathogen is contained can be judged according to a fluorescence signal and an amplification curve in the amplification product. When the quantitative fluorescent PCR is used as a detection means, the specific method for the characterization is not limited, and may be, for example, a dye method or a probe method commonly used in the art. Among them, the probe method is preferred to the dye method in terms of specificity, and thus the probe method is preferably used for the characterization. In some preferred embodiments, the primer set provided by the invention further comprises a probe for detecting feline herpes type1 virus, a probe for detecting feline calicivirus, a probe for detecting mycoplasma, a probe for detecting chlamydia felis and a probe for detecting bordetella bronchiseptica.

Specifically, the probe for detecting the feline herpes type1 virus has a nucleotide sequence shown as SEQ ID NO.11, or a nucleotide sequence with at least 85% of identity with SEQ ID NO. 11;

The probes in the embodiment are designed through highly conserved regions of various pathogens, have no cross reaction with genes of other pathogens, and have the advantages of strong specificity and high sensitivity. Similarly, "identity" refers to a nucleotide sequence that includes at least 85% (e.g., but not limited to, 85%, 88%, 90%, 92%, 95%, 98%, 99% or more) identity to the nucleotide sequence set forth in SEQ ID Nos. 11-15 described in the present embodiment and has the same function. When the nucleotide sequences shown in SEQ ID Nos. 11 to 15 are selected as the probes provided in the present embodiment, they have greater specificity and greater sensitivity.

In some preferred embodiments, both ends of the probe for detecting feline herpes type1 virus, the probe for detecting feline calicivirus, the probe for detecting mycoplasma, the probe for detecting feline chlamydia and the probe for detecting bordetella bronchiseptica independently contain a fluorescence quenching group and a fluorescence reporter group different from each other. In this embodiment, the specific probe does not have to include a fluorescence reporter group and a fluorescence quencher group, and any group can be used to connect to the probe and perform the corresponding characterization function.

Preferably, the fluorescent reporter group comprises FAM, HEX, ROX, CY5 or CY 5.5. In this embodiment, the correspondence between the probe and each of the fluorophores is not limited, as long as each probe is ensured to contain a different fluorophore. The probe for detecting feline herpes type1 virus preferably comprises FAM, the probe for detecting feline calicivirus preferably comprises HEX, the probe for detecting mycoplasma preferably comprises ROX, the probe for detecting feline chlamydia preferably comprises CY5, and the probe for detecting bordetella bronchiseptica preferably comprises CY 5.5.

Preferably, the fluorescence quenching group comprises BHQ1, BHQ2 or BHQ 3.

In some preferred embodiments, the primers for detecting feline herpes type1 virus, the primers for detecting feline calicivirus, the primers for detecting mycoplasma, the primers for detecting feline chlamydia, and the primers for detecting bordetella bronchiseptica work at a concentration of 5-20nmol, for example, but not limited to, 5nmol, 8nmol, 10nmol, 12nmol, 15nmol, 18nmol, or 20 nmol.

Preferably, the working concentration of the probe for detecting feline herpes type1 virus, the probe for detecting feline calicivirus, the probe for detecting mycoplasma, the probe for detecting feline chlamydia and the probe for detecting bordetella bronchiseptica is 1-10nmol, for example, but not limited to, 1nmol, 2nmol, 3nmol, 5nmol, 8nmol or 10 nmol.

By limiting the working concentration of the primer and the probe, the primer or the probe is prevented from being degraded due to too low working concentration, primer dimer due to too high concentration is also prevented, and meanwhile, the maximum utilization of the primer or the probe can be ensured within the optimal working concentration range, so that waste is avoided.

Based on the same inventive concept as the primer set provided by the invention, the invention also provides a product for detecting the feline respiratory pathogens, wherein the product comprises the primer set, so that the product for detecting the feline respiratory pathogens provided by the invention has the overall beneficial effects as the primer set provided by the invention, and the details are not repeated.

Preferably, the product comprises a reagent or kit.

In some preferred embodiments, the kit further comprises at least one of a PCR reaction solution, a positive control, or a negative control.

The PCR reaction solution preferably contains the primer set provided by the invention, the PCR reaction solution containing the primer set provided by the invention is packaged in advance, only extracted genomic nucleic acid needs to be added when the PCR reaction solution is used, the operation is simple and convenient, and the volume of the packaged PCR reaction solution is preferably 20 mu L per reaction.

Preferably, the positive control comprises a plasmid containing part or all of the feline herpes type1 virus gene sequence, a plasmid containing part or all of the feline calicivirus gene sequence, a plasmid containing part or all of the mycoplasma gene sequence, a plasmid containing part or all of the feline chlamydia gene sequence, and a plasmid containing part or all of the bordetella bronchiseptica gene sequence; plasmids containing part or all of the pathogen gene sequence can be used as the positive control in this embodiment, and plasmids containing part of the gene sequence are preferably used to save costs.

Preferably, the negative control comprises sterilized deionized water.

By setting the positive control substance and the negative control substance, false negative or false positive results can be effectively avoided, and the detection accuracy is further ensured.

According to the third aspect of the present invention, there is also provided a method for detecting a feline respiratory pathogen for non-diagnostic purposes, wherein a genomic nucleic acid of a sample to be detected is used as a template, a PCR reaction is performed using the primer set or the product, and whether the sample to be detected contains a feline respiratory pathogen is determined according to a specific fragment or an amplification curve contained in an amplification product:

It should be noted that, because the primer set provided by the present invention can be used for different types of PCR reactions, there are corresponding result determination methods for the different types of PCR reactions applied. Typically, when conventional PCR is used, it is determined whether the test sample contains herpesvirus felis 1, calicivirus felis, mycoplasma, chlamydia felis, or bordetella bronchiseptica, or which of the above pathogens is contained, based on the presence or absence of a band of the amplification product and the size of the fragment of the amplification product. Typically, when the fluorescent quantitative PCR detection is performed, whether the sample to be detected contains the herpesvirus felis 1, the calicivirus felis, the mycoplasma, the chlamydia felis or the bordetella bronchiseptica or which of the pathogens is contained can be judged according to the fluorescent signal and the amplification curve in the amplification product. Different PCR reaction types and corresponding different result determination methods can be matched with the primer group or the kit provided by the invention to realize accurate molecular identification of the feline respiratory pathogens.

Furthermore, it is particularly emphasized that the method for detecting feline respiratory pathogens provided by the present invention is a method for non-disease diagnostic or therapeutic purposes, and is only useful, for example, for the detection or identification of laboratory samples.

In some preferred embodiments, the PCR reaction is a fluorescent quantitative PCR reaction. The fluorescent quantitative PCR technology has the advantages of rapidness, sensitivity, specificity and simplicity, and in addition, the multiple fluorescent quantitative PCR method can also obviously improve the defects of long time consumption and low flux of single pathogen detection.

In the embodiment, the reaction conditions of the fluorescent quantitative PCR are further adjusted and optimized, so that the specificity of the identification reaction performed under the conditions is stronger, and the result accuracy is higher.

In some preferred embodiments, the Ct value of each channel of the positive control is less than 30, preferably 25 ± 2; and each channel of the negative control was not amplified.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The main reagent information used in the examples of the present invention is as follows:

example 1

1 primer Probe

1.1 design and screening of primer probes

The inventor downloads a large number of gene sequences of herpesvirus catarrhalis type1, feline calicivirus, mycoplasma, feline chlamydia and bordetella bronchiseptica respectively in GenBank, finds out corresponding primer probes designed in the highly conserved regions of the above 5 pathogens respectively through sequence comparison, designs at least two groups of primers and one probe for each pathogen, and screens out primer groups and probes suitable for multiple detection of feline respiratory pathogens and combinations thereof through experimental comparison verification of the performance of the primer probes, wherein the sequences are specifically as follows:

SEQ ID NO：1：5’-CAAGTATGACGGCTTACGATC-3’；

SEQ ID NO：2：5’-GTTGGGTCTTATGTGCCAGTC-3’；

SEQ ID NO：3：5’-TCAGCGTTGTTGTCACAGTTCTA-3’；

SEQ ID NO：4：5’-GCTTAAGATACCCAATCTCGTA-3’；

SEQ ID NO：5：5’-GCTTACAGCTATTAGTTCACT-3’；

SEQ ID NO：6：5’-TGTTGTCACAGTTAAGAGGAAC-3’；

SEQ ID NO：7：5’-TAGGTGCTGTAGCCAGGAT-3’；

SEQ ID NO：8：5’-AAGCTACATGGGCAAGAGCTG-3’；

SEQ ID NO：9：5’-ATGACACAGGTGCAGGGTTG-3’；

SEQ ID NO：10：5’-TTGACACAGAGCTGGCCACTG-3’；

SEQ ID NO：11：5’-FAM-TTCCAATCGTAGGAACTTCAACGGA-BHQ1-3’；

SEQ ID NO：12：5’-HEX-AACGCAGATGACCAGACGCATGACC-BHQ1-3’；

SEQ ID NO：13：5’-ROX-ACTTGCGATGCAGATTGGAGTGCTGAG-BHQ2-3’；

SEQ ID NO：14：5’-CY5-CAGTCGCAACATACAGTGTCAATTCC-BHQ3-3’；

SEQ ID NO：15：5’-CY5.5-TTCACAGACGGAATATGAGGCTAC-BHQ3-3’；

SEQ ID NO：16：5’-ACTGCGGTGAGGTGAAGTTGC-3’；

SEQ ID NO：17：5’-CTTCAGGATCTACTGTCGTGCGT-3’；

SEQ ID NO：18：5’-GTCACAATTTGAAATCAATCG-3’；

SEQ ID NO：19：5’-TGAACAATGCATCACTCCTCAA-3’；

SEQ ID NO：20：5’-GGATTTATAAGAGTAAATATAG-3’；

SEQ ID NO：21：5’-TCTATATCCAAAATTCTATGCG-3’；

SEQ ID NO：22：5’-CGATGAGGAAGCTAATGCA-3’；

SEQ ID NO：23：5’-TCATCGACTCTATGTGTCCA-3’；

SEQ ID NO：24：5’-ACGAGGATCCCAGAGAATCT-3’；

SEQ ID NO：25：5’-CAACAAATCGAGGGATACGTA-3’。

wherein, the upstream and downstream primers and probe sequences for detecting the feline herpes type1 virus are respectively SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 11, SEQ ID NO: 11 are labeled FAM and BHQ1 at the 5 'and 3' ends, respectively;

the sequences of the upstream primer and the downstream primer for detecting the feline calicivirus and the probe are respectively SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 18. SEQ ID NO: 19 and SEQ ID NO: 12, SEQ ID NO: the 5 'and 3' ends of 12 are labeled HEX and BHQ1, respectively;

the sequences of the upstream primer and the downstream primer for detecting mycoplasma are respectively SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 20. SEQ ID NO: 21 and SEQ ID NO: 13, SEQ ID NO: the 5 'and 3' ends of 13 are labeled as ROX and BHQ2, respectively;

the sequences of an upstream primer and a downstream primer and a probe for detecting the cat chlamydia are respectively SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 22. SEQ ID NO: 23 and SEQ ID NO: 14, SEQ ID NO: the 5 'and 3' ends of 14 are labeled CY5 and BHQ3, respectively;

the upstream and downstream primers and probe sequences for detecting bordetella bronchiseptica are respectively SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 24. SEQ ID NO: 25 and SEQ ID NO: 15, SEQ ID NO: 15 are labeled CY5.5 and BHQ3, respectively.

2 preparation of Positive control

After the corresponding strains are expanded and cultured, the steps of the specification of a Biospin plasmid DNA miniprep kit of Hangzhou Bosun science and technology Limited are used for extracting positive plasmids, the extracted positive plasmids are respectively named as pFVH-1, pFVV, pMyc, pF-Chl and pBb, pFVH-1, pFVV, pMyc, pF-Chl and pBb are mixed according to the proportion of 1:1:1:1: 1:1, the obtained positive mixed liquid is subjected to fluorescence PCR amplification by using SEQ ID NO: 1-SEQ ID NO: 15, when the Ct value of each channel is 25 +/-2, the positive plasmids can be used as standby products and are stored in the mixed liquid at the temperature of 500-20 ℃ by subpackaging, wherein the positive mixed liquid is obtained.

3 preparation of negative control

The negative control was sterilized deionized water, and each tube was filled with 500. mu. L and stored at-20 ℃ for further use.

4 finished product Assembly of the kit

4.1 fluorescent PCR reaction solution

The kit comprises a primer and a probe for specifically detecting the feline herpesvirus type1, a primer and a probe for specifically detecting the feline calicivirus, a primer and a probe for specifically detecting the mycoplasma, a primer and a probe for specifically detecting the feline chlamydia and a primer and a probe for specifically detecting the bordetella bronchiseptica, and the fluorescent PCR reaction solution is subpackaged into PCR tubes according to the required dosage of each reaction in advance.

4.2 Positive control

Contains pFV-1, pFCV, pMyc, pF-Chl and pBb. Positive plasmid solutions prepared by mixing pFVH-1, pFCV, pMyc, pF-Chl and pBb at a ratio of 1:1:1:1:1 were tested using the fluorescent PCR reaction solution of 4.1, and the Ct values of the respective channels were 25. + -. 2 (results are shown in FIG. 1).

4.3 negative control

The negative control was sterilized deionized water, and the fluorescence PCR reaction solution from 4.1 was used for detection, with no amplification in each channel.

4.4 description

The kit contains a specification which comprises kit components, a use method, result judgment and the like.

4.5 external packing

The external package is packaged by using a specific packaging material and a specific packaging mode of PCR products of Hangzhou Bori science and technology limited company.

Example 2 detection of Cat respiratory tract samples in Pet Hospital after Collection

1 Experimental procedures

1.1 sample Source

A pet hospital is provided with a cat disease detection room, and part of samples are collected for detecting respiratory pathogens.

1.2 sample handling

Sample nucleic acid was extracted using a Simply P virus DNA/RNA extraction kit from Hangzhou Bori science and technology, Inc., with reference to the kit instructions.

1.3 amplification reagent preparation

Taking out a corresponding number of PCR tubes containing fluorescent PCR reaction liquid according to the number of samples to be detected, using a palm centrifuge for instantaneous centrifugation after melting to concentrate reagents at the tube bottom, opening the PCR tube cover, sequentially adding a positive and negative reference substance, a positive reference substance and 5 mu L of nucleic acid to be detected extracted from 2 into different PCR tubes, wherein the volume of liquid in each reaction tube is 25 mu L, repeating the steps once for each sample, covering the PCR tube cover, and performing PCR amplification in a L ineGene9600Plus PCR instrument of Hangzhou Bori science and technology Limited after air bubbles are removed through instantaneous centrifugation.

1.4PCR reaction conditions

The conditions of the PCR amplification reaction are as follows: 50 ℃ for 10 min; pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, for 40 cycles; the fluorescence signal of each channel was collected at 60 ℃.

2 analysis of results

2.1 validity of the results

In the same experiment, the Ct values of all channels of positive control products FAM, HEX, ROX, CY5 and CY5.5 are all less than 30, all channels of the negative control products are not amplified, the experiment is effective, and otherwise, the experiment is invalid.

2.2 principle of determination of results

If the FAM channel can be amplified, the cat to be detected is infected with the cat herpesvirus type1, namely the cat herpesvirus type1 nucleic acid is positive; if the HEX channel can be amplified, the fact that the cat to be detected is infected with the feline calicivirus is indicated, namely the feline calicivirus nucleic acid is positive; if the ROX channel can be amplified, the condition that the cat to be detected is infected with mycoplasma is indicated, namely the mycoplasma nucleic acid is positive; if the CY5 channel can be amplified, the cat to be detected is infected with the cat chlamydia, namely the cat chlamydia nucleic acid is positive; if the CY5.5 channel can be amplified, the condition that the cat to be detected is infected with the bordetella bronchiseptica is indicated. When all channels were not amplified, the cat to be tested was not infected with the above 5 pathogens.

2.3 summary of test results

Of 10 samples, 3 cat herpesvirus positive samples, 1 cat calicivirus positive sample, 2 mycoplasma positive samples, 1 cat chlamydia positive sample, 0 bordetella bronchiseptica and 4 negative pathogens of the above 5 types were detected. Among them, 1 sample had mixed infection with pathogens (the detection result was positive for the presence of two or more pathogens). The detailed results are summarized in the following table.

TABLE 1 summary of sample test results

Wherein "- -" indicates that the detection result has no Ct value.

Example 3 specificity verification experiment

The feline respiratory pathogen detection method described in example 1 was followed to detect feline herpesvirus type1, feline calicivirus, mycoplasma, feline chlamydia, bordetella bronchiseptica, canine herpesvirus, feline parvovirus, feline coronavirus, toxoplasma, haemophilus, positive plasmid pFHV-1, positive plasmid pFCV, positive plasmid pMyc, positive plasmid pF-Chl, positive plasmid pBb, positive control, negative control, and analyze the results for positive and negative controls, indicating that the experiment was valid. The detection kit and the detection method have the advantages that only FAM channels of the feline herpesvirus type1 and the positive plasmid pFV-1 have Ct values, only HEX channels of the feline calicivirus and the positive plasmid pFCV have Ct values, only ROX channels of the mycoplasma and the positive plasmid pMyc have Ct values, only CY5 channels of the feline chlamydia and the positive plasmid pF-Chl have Ct values, only CY5.5 channels of the bordetella bronchiseptica and the positive plasmid pBb have Ct values, and no amplification exists in samples containing other viruses (the result is shown in figure 2).

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Hangzhou Bori science and technology Co., Ltd

<120> primer group, product, method and application for detecting feline respiratory pathogens

<130>2020

<160>25

<170>PatentIn version 3.5

<210>1

<211>21

<212>DNA

<213> Artificial sequence

<400>1

caagtatgac ggcttacgat c 21

<210>2

<211>21

<212>DNA

<213> Artificial sequence

<400>2

gttgggtctt atgtgccagt c 21

<210>3

<211>23

<212>DNA

<213> Artificial sequence

<400>3

tcagcgttgt tgtcacagtt cta 23

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<400>4

gcttaagata cccaatctcg ta 22

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<400>5

gcttacagct attagttcac t 21

<210>6

<211>22

<212>DNA

<213> Artificial sequence

<400>6

tgttgtcaca gttaagagga ac 22

<210>7

<211>19

<212>DNA

<213> Artificial sequence

<400>7

taggtgctgt agccaggat 19

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<400>8

aagctacatg ggcaagagct g 21

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<400>9

atgacacagg tgcagggttg 20

<210>10

<211>21

<212>DNA

<213> Artificial sequence

<400>10

ttgacacaga gctggccact g 21

<210>11

<211>25

<212>DNA

<213> Artificial sequence

<400>11

ttccaatcgt aggaacttca acgga 25

<210>12

<211>25

<212>DNA

<213> Artificial sequence

<400>12

aacgcagatg accagacgca tgacc 25

<210>13

<211>27

<212>DNA

<213> Artificial sequence

<400>13

acttgcgatg cagattggag tgctgag 27

<210>14

<211>26

<212>DNA

<213> Artificial sequence

<400>14

cagtcgcaac atacagtgtc aattcc 26

<210>15

<211>24

<212>DNA

<213> Artificial sequence

<400>15

ttcacagacg gaatatgagg ctac 24

<210>16

<211>21

<212>DNA

<213> Artificial sequence

<400>16

actgcggtga ggtgaagttg c 21

<210>17

<211>23

<212>DNA

<213> Artificial sequence

<400>17

cttcaggatc tactgtcgtg cgt 23

<210>18

<211>21

<212>DNA

<213> Artificial sequence

<400>18

gtcacaattt gaaatcaatc g 21

<210>19

<211>22

<212>DNA

<213> Artificial sequence

<400>19

tgaacaatgc atcactcctc aa 22

<210>20

<211>22

<212>DNA

<213> Artificial sequence

<400>20

ggatttataa gagtaaatat ag 22

<210>21

<211>22

<212>DNA

<213> Artificial sequence

<400>21

tctatatcca aaattctatg cg 22

<210>22

<211>19

<212>DNA

<213> Artificial sequence

<400>22

cgatgaggaa gctaatgca 19

<210>23

<211>20

<212>DNA

<213> Artificial sequence

<400>23

tcatcgactc tatgtgtcca 20

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<400>24

acgaggatcc cagagaatct 20

<210>25

<211>21

<212>DNA

<213> Artificial sequence

<400>25

caacaaatcg agggatacgt a 21

Claims

1. A primer group for detecting feline respiratory pathogens, which is characterized by comprising a primer for detecting feline herpes type1 virus, a primer for detecting feline calicivirus, a primer for detecting mycoplasma, a primer for detecting feline chlamydia and a primer for detecting bordetella bronchiseptica;

2. The primer set according to claim 1, further comprising a probe for detecting feline herpes type1 virus, a probe for detecting feline calicivirus, a probe for detecting mycoplasma, a probe for detecting chlamydia felis, and a probe for detecting bordetella bronchiseptica;

the probe for detecting the Chlamydia felis has a nucleotide sequence shown as SEQ ID NO.14 or a nucleotide sequence with at least 85% of identity with SEQ ID NO. 14;

3. The primer set according to claim 2, wherein both ends of the probe for detecting feline herpes type1 virus, the probe for detecting feline calicivirus, the probe for detecting mycoplasma, the probe for detecting feline chlamydia and the probe for detecting bordetella bronchiseptica independently contain a fluorescence quenching group and a fluorescence reporter group different from each other, respectively;

preferably, the fluorescence quenching group comprises BHQ1, BHQ2 or BHQ 3.

4. The primer set according to any one of claims 1 to 3, wherein the working concentration of the primer for detecting feline herpes type1 virus, the primer for detecting feline calicivirus, the primer for detecting mycoplasma, the primer for detecting feline chlamydia and the primer for detecting Bordetella bronchiseptica is 5 to 20 nmol;

5. A product for detecting a feline respiratory pathogen comprising the primer set of any one of claims 1-4;

preferably, the product comprises a reagent or kit.

6. The product of claim 5, wherein the kit further comprises at least one of a PCR reaction solution, a positive control, or a negative control;

preferably, the positive control comprises a plasmid containing part or all of the feline herpes type1 virus gene sequence, a plasmid containing part or all of the feline calicivirus gene sequence, a plasmid containing part or all of the mycoplasma gene sequence, a plasmid containing part or all of the feline chlamydia gene sequence, and a plasmid containing part or all of the bordetella bronchiseptica gene sequence;

7. A method for detecting a feline respiratory pathogen for non-diagnostic purposes, which comprises performing a PCR reaction using a genomic nucleic acid of a test sample as a template using the primer set of any one of claims 1 to 4 or the product of claim 5 or 6, and determining whether the test sample contains a feline respiratory pathogen based on a specific fragment or amplification curve contained in the amplification product:

8. The method of claim 7, wherein the PCR reaction is a fluorescent quantitative PCR reaction;

9. The method of claim 8, wherein the Ct value of each channel of the positive control is less than 30, preferably 25 ± 2;

10. Use of a primer set according to any one of claims 1 to 4, a product according to claim 5 or 6 or a method according to any one of claims 7 to 9 for the detection of a feline respiratory pathogen.