Primer probe combination capable of simultaneously detecting nucleic acids of six pathogens of alimentary canal and respiratory tract of dog and application of primer probe combination

Abstract

The invention provides a primer and a fluorescent probe combination capable of simultaneously detecting nucleic acids of six pathogens of a dog and an application of the primer and the fluorescent probe combination. The primer and fluorescent probe combination comprises a group of primer probe combination for detecting three dog digestive tract pathogens, namely canine parvovirus, canine bordetella and canine adenovirus, and a group of primer probe combination for detecting three dog respiratory tract pathogens, namely canine distemper virus, canine coronavirus and canine parainfluenza virus. The invention also provides a six-linked fluorescent RT-qPCR detection method and a kit using the primer probe combination. According to the method, one group of primer probe combination can be selected for detection, or two groups of primer probe combinations can be selected for detection at the same time. According to the method disclosed by the invention, the signal intensity amplified by each three channels of two tubes is monitored and output in real time, and qualitative analysis of common canine pathogen detection is realized. Operation is convenient and fast, sensitivity is high, and cost is low.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pathogen detection in the dog breeding industry, and particularly relates to a primer probe combination for simultaneously detecting nucleic acids of six pathogens in the digestive tract and respiratory tract of dogs and application thereof. TECHNICAL BACKGROUND

[0002] Common canine animal epidemic diseases mainly focus on six pathogens related to the digestive tract and respiratory tract, such as canine parvovirus (CPV), canine Bordetella (Bb), canine adenovirus (CAV), canine distemper virus (CDV), canine coronavirus (CoV) and canine parainfluenza virus (CPIV). CPV mainly causes acute hemorrhagic gastroenteritis and acute myocarditis in puppies. Bordetella bronchiseptica (Bb) is a gram-negative small coccus, which mainly causes cough, paroxysmal inspiratory dyspnea, body temperature rises to 39-40℃, and is accompanied by tonsillar and pharyngeal inflammation. CAV-II type causes canine contact respiratory disease related to respiratory disease and enteritis, but does not cause pneumonia symptoms, and can cause puppy cough, also known as kennel cough. CDV infection causes canine distemper, mainly including respiratory type, digestive type, neurological type and mixed type, and CDV infection can cause systemic immunosuppression with a high mortality rate. CPIV infection mainly causes respiratory disease in dogs, and is one of the important pathogens of canine infectious respiratory system disease.

[0003] Chinese patent application CN 110846438 A discloses a quadruple real-time fluorescent quantitative PCR detection of canine adenovirus type II, canine distemper virus, canine parvovirus and canine parainfluenza virus, and provides a primer and probe set for the real-time fluorescent quantitative PCR detection, and based on the primer and probe set, provides a kit for simultaneously identifying and detecting canine adenovirus type II, canine distemper virus, canine parvovirus and canine parainfluenza virus.

[0004] Chinese patent application CN 116287457 A discloses a primer combination for differential diagnosis of canine distemper virus, canine adenovirus, canine parainfluenza virus and Bordetella, and application thereof, comprising: outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. The present application can be used for differential diagnosis of canine distemper virus pathogen genes, canine adenovirus pathogen genes, canine parainfluenza virus pathogen genes and Bordetella bronchiseptica pathogen genes causing canine respiratory disease.

[0005] At present, there is no detection method or kit for simultaneously detecting three kinds of canine digestive tract pathogens, i.e., canine parvovirus, canine Bordetella and canine adenovirus, simultaneously detecting three kinds of canine respiratory tract pathogens, i.e., canine distemper virus, canine coronavirus and canine parainfluenza virus, and simultaneously detecting six kinds of canine pathogens, i.e., canine parvovirus, canine Bordetella, canine adenovirus, canine distemper, canine coronavirus and canine parainfluenza, which is convenient, fast, high in sensitivity and low in cost. SUMMARY

[0006] Therefore, the present application provides an RT-qPCR detection primer and fluorescent probe combination for simultaneously detecting three kinds of canine digestive tract pathogens, i.e., canine parvovirus, canine Bordetella and canine adenovirus, which contains a forward primer CPV-F, a reverse primer CPV-R and a FAM-labeled fluorescent probe CPV-P of a canine parvovirus VP2 gene.

[0007] The present application also provides an RT-qPCR detection primer and fluorescent probe combination for simultaneously detecting three kinds of canine respiratory tract pathogens, i.e., canine distemper virus, canine coronavirus and canine parainfluenza virus, which contains a forward primer CDV-F, a reverse primer CDV-R and a FAM-labeled fluorescent probe CDV-P of a canine distemper virus N protein gene, a forward primer COV-F, a reverse primer COV-R and a VIC-labeled fluorescent probe COV-P of a canine coronavirus ORF1 gene, and a forward primer CPIV-F, a reverse primer CPIV-R and a Texas Red-labeled fluorescent probe CPIV-P of a canine parainfluenza virus N protein gene.

[0008] Further, the nucleotide of the CPV-F is shown in the sequence SEQ ID NO. 1,

[0009] the nucleotide of the CPV-P is shown in the sequence SEQ ID NO. 2,

[0010] the nucleotide of the CPV-R is shown in the sequence SEQ ID NO. 3;

[0011] the nucleotide of the Bb-F is shown in the sequence SEQ ID NO. 4,

[0012] the nucleotide of the Bb-P is shown in the sequence SEQ ID NO. 5,

[0013] The nucleotide of the Bb-R is shown in sequence SEQ ID NO. 6;

[0014] The nucleotide of the CAV-F is shown in sequence SEQ ID NO. 7,

[0015] The nucleotide of the CAV-P is shown in sequence SEQ ID NO. 8,

[0016] The nucleotide of the CAV-R is shown in sequence SEQ ID NO. 9.

[0017] Further, the nucleotide of the CDV-F is shown in sequence SEQ ID NO. 10,

[0018] The nucleotide of the CDV-P is shown in sequence SEQ ID NO. 11,

[0019] The nucleotide of the CDV-R is shown in sequence SEQ ID NO. 12;

[0020] The nucleotide of the COV-F is shown in sequence SEQ ID NO. 13,

[0021] The nucleotide of the COV-P is shown in sequence SEQ ID NO. 14,

[0022] The nucleotide of the COV-R is shown in sequence SEQ ID NO. 15;

[0023] The nucleotide of the CPIV-F is shown in sequence SEQ ID NO. 16,

[0024] The nucleotide of the CPIV-P is shown in sequence SEQ ID NO. 17,

[0025] The nucleotide of the CPIV-R is shown in sequence SEQ ID NO. 18.

[0026] The application also provides a RT-qPCR detection primer, fluorescent probe combination for simultaneously detecting six kinds of canine pathogens of canine parvovirus, canine Bordetella, canine adenovirus, canine distemper virus, canine coronavirus and canine parainfluenza virus, which comprises two groups of RT-qPCR detection primers, fluorescent probe combinations, which are the above-mentioned three kinds of canine digestive tract pathogen RT-qPCR detection primers, fluorescent probe combinations and the above-mentioned three kinds of canine respiratory tract pathogen RT-qPCR detection primers, fluorescent probe combinations respectively.

[0027] The application also provides a six-union fluorescent RT-qPCR detection method for canine digestive tract and respiratory tract pathogen nucleic acid, which comprises the following steps:

[0028] 1) Sample nucleic acid extraction;

[0029] 2) Calculate the number of reactions required for the experiment n = number of negative controls + number of positive controls + number of samples;

[0030] 3) Each of the number of reactions is respectively configured with K1 and K2 two-hole reagents, the K1 hole reagent is combined with the RT-qPCR reaction liquid prepared by the three kinds of canine digestive tract pathogen RT-qPCR detection primers and fluorescent probes, and the K2 hole reagent is combined with the RT-qPCR reaction liquid prepared by the three kinds of canine respiratory tract pathogen RT-qPCR detection primers and fluorescent probes; The prepared reagents are respectively added to the corresponding K1 hole and K2 hole PCR reaction tubes;

[0031] 4) The K1 hole and the K2 hole are respectively added with sample nucleic acid templates, positive controls and negative controls, and simultaneously and under the same conditions, RT-qPCR reactions are carried out;

[0032] 5) The K1 hole and the K2 hole are selected to collect fluorescence signals to obtain Ct values in three fluorescence channels of FAM, VIC and Texas Red; The FAM positive signal in the K1 hole can detect canine parvovirus, the VIC positive signal in the K1 hole can detect canine Bordetella, and the Texas Red positive signal in the K1 hole can detect canine adenovirus; The FAM positive signal in the K2 hole can detect canine distemper virus, the VIC positive signal in the K2 hole can detect canine coronavirus, and the Texas Red positive signal in the K2 hole can detect canine parainfluenza virus.

[0033] Further, the reaction conditions for simultaneously carrying out PCR amplification in the K1 hole and the K2 hole are as follows: reverse transcription 50 DEG C: 10 min, 1 cycle; pre-denaturation 95 DEG C: 30 s, 1 cycle; PCR 95 DEG C: 10 s, 40 cycles.

[0034] Further, the Ct value obtained is ≤35 and has obvious exponential growth, which is judged as a positive signal; the Ct value obtained is in the range of 35-38, and after repeated detection of the sample, the Ct value is still in the range of 35-38 and has obvious exponential growth, which is judged as a positive signal, otherwise it is a negative signal; the Ct value obtained is > 38 or has no Ct value, the line is a straight line or a slight slant line and has no obvious exponential growth, which is judged as a negative signal.

[0035] The application also provides a canine digestive tract and respiratory tract pathogen nucleic acid six-union fluorescent RT-qPCR detection kit, the kit comprises RT-qPCR reaction liquid, enzyme mixed solution, negative quality control, positive quality control, wherein the RT-qPCR reaction liquid comprises two groups of RT-qPCR reaction liquids prepared respectively, one group contains three kinds of canine digestive tract pathogen RT-qPCR detection primers and fluorescent probe combination, and the other group contains three kinds of canine respiratory tract pathogen RT-qPCR detection primers and fluorescent probe combination.

[0036] The application also provides a use method of the above-mentioned canine digestive tract and respiratory tract pathogenic nucleic acid six-universal fluorescent RT-qPCR detection kit. After each of the detection primer and fluorescent probe combinations in each group completes the fluorescent RT-qPCR of the detection sample in the same PCR reaction system, the Ct value is obtained through the FAM, VIC and Texas Red fluorescent detection channels respectively; and the two groups of RT-qPCR detection primer and fluorescent probe combination reaction systems perform the RT-qPCR reaction at the same time and under the same conditions.

[0037] The application has the following beneficial effects:

[0038] 1. The kit or method provided by the application can select one group of reaction systems for detection, or two groups of reaction systems for detection at the same time. Therefore, the detection method or kit for detecting canine parvovirus, canine bordetella and canine adenovirus, three canine digestive tract pathogens, three canine respiratory tract pathogens, canine distemper virus, canine coronavirus and canine parainfluenza virus, and six canine pathogens, canine parvovirus, canine bordetella, canine adenovirus, canine distemper, canine coronavirus and canine parainfluenza, can be detected at the same time, and the operation is convenient, fast, sensitive and low in cost.

[0039] 2. The application uses fluorescent RT-PCR technology to simultaneously detect three pathogens (canine parvovirus, canine bordetella and canine adenovirus) commonly causing canine digestive tract diseases and three pathogens (canine distemper, canine coronavirus and canine parainfluenza) commonly causing respiratory diseases, a total of six pathogens. The canine parvovirus VP2 gene, the FIM2 gene of canine bordetella, the S protein of canine adenovirus, the N protein of canine distemper virus, the ORF1 gene of canine coronavirus and the N protein of canine parainfluenza virus are selected, specific primers and probes are designed in the highly conserved regions of these genes, three channels are amplified in two tubes, the signal intensity of the corresponding channels in the PCR process is monitored and output by the instrument in real time, and qualitative analysis of canine common digestive tract and respiratory tract pathogen detection is realized. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a FAM channel linear experimental data graph for canine parvovirus detection of the application.

[0041] Figure 2 It is a FAM channel standard curve schematic diagram for canine parvovirus detection of the application.

[0042] Figure 3 It is an experimental data graph of the detection limit of the canine parvovirus detection kit of the application.

[0043] Figure 4 It is a specific experimental data graph of the canine parvovirus detection kit of the application.

[0044] Figure 5This is a graph showing the linear experimental data of the VIC channel for the detection of Bordetella canis according to the present invention.

[0045] Figure 6 This is a schematic diagram of the VIC channel standard curve for the detection of Bordetella canis according to the present invention.

[0046] Figure 7 This is a graph showing the experimental data for the detection limit of Bordetella canis in the detection kit of this invention.

[0047] Figure 8 This is a graph showing the specificity experimental data of the detection kit for Bordetella canis in this invention.

[0048] Figure 9 This is a graph showing the linear experimental data of the Texas Red channel for canine adenovirus detection in this invention.

[0049] Figure 10 This is a schematic diagram of the Texas Red channel standard curve for canine adenovirus detection in this invention.

[0050] Figure 11 This is a graph showing the experimental data for the detection limit of canine adenovirus in the detection kit of this invention.

[0051] Figure 12 This is a graph showing the specificity of the canine adenovirus detection kit of this invention.

[0052] Figure 13 This is a graph showing the linear experimental data of the FAM channel for canine distemper virus detection in this invention.

[0053] Figure 14 This is a schematic diagram of the FAM channel standard curve for canine distemper virus detection in this invention.

[0054] Figure 15 This is a graph showing the experimental data for the detection limit of canine distemper virus in the detection kit of this invention.

[0055] Figure 16 This is a graph showing the specificity of the canine distemper virus detection kit of the present invention.

[0056] Figure 17 This is a graph showing the linear experimental data of the VIC channel for canine coronavirus detection in this invention.

[0057] Figure 18 This is a schematic diagram of the VIC channel standard curve for canine coronavirus detection in this invention.

[0058] Figure 19 This is a graph showing the experimental data for the detection limit of canine coronavirus in the detection kit of this invention.

[0059] Figure 20 This is a graph showing the specificity of the detection kit for canine coronavirus in this invention.

[0060] Figure 21 Texas Red channel linear experimental data graph for detection of canine parainfluenza virus of the application.

[0061] Figure 22 Texas Red channel standard curve schematic diagram for detection of canine parainfluenza virus of the application.

[0062] Figure 23 Detection limit experimental data graph for detection of canine parainfluenza virus of the application.

[0063] Figure 24 Specificity experimental data graph for detection of canine parainfluenza virus of the application. DETAILED DESCRIPTION

[0064] Example 1: RT-qPCR detection primer and fluorescent probe combination of the application, and related reagents, instruments, product related parameter description

[0065] 1. Reagents and materials

[0066] Reagents: All reagents used in the experiment are analytical pure, and the experimental water should meet the requirements of first-class water in GB / T 6682.

[0067] Nucleic acid extraction kit, Rnase free water, qPCR Probe Master Mix.

[0068] Positive control: positive plasmid containing the target fragment.

[0069] Negative control: Rnase free water.

[0070] 2. Consumables

[0071] Centrifuge tube, 0.1ml / 0.2mL sterile RNase-free PCR reaction tube (according to qPCR instrument), 1.5mL sterile RNase-free centrifuge tube.

[0072] RNase-free gun head: 10μL, 200μL, 1000μL.

[0073] 3. RT-qPCR detection primer and fluorescent probe combination of the application

[0074] Table 1 qPCR primer sequence and concentration

[0075]

[0076]

[0077] 4. Instruments

[0078] High speed refrigerated centrifuge, 12000r / min or above. Adjustable pipettor, maximum range of 10μL, 200μL, 1000μL. Refrigerator, -20℃ Multi-channel fluorescent quantitative qPCR amplifier.

[0079] Sample: The collection and processing of samples shall comply with the provisions of GB / T 36871.

[0080] 5qPCR reaction system (Table 2)

[0081]

[0082]

[0083] Example 2: Kit composition and use method of the application

[0084] The kit of the application is designed for highly conserved regions of six pathogens of canine parvovirus, canine distemper virus, canine adenovirus, canine parainfluenza virus, canine coronavirus and canine bronchial septic Bordetella, specific primers and probes, two tubes of six joint detection, in the presence of target gene template in the reaction system, PCR reaction can be carried out and release fluorescence signal. The instrument is used to monitor and output the signal intensity of the corresponding channel in the PCR process, and the qualitative analysis of the detection result is realized.

[0085] The main components of the kit are shown in Table 3:

[0086] Table 3:

[0087] Component 50 reaction Reaction solution Al 1 tube (500 μL) Reaction solution Bl 1 tube (500 μL) Reaction solution A2 1 tube (500 μL) Reaction solution B2 1 tube (500 μL) Mixed enzyme solution 1 tube (100 μL) Negative control 1 tube (100 μL) Positive control 1 tube (100 μL)

[0088] The applicable detection instrument is:

[0089] ABI 7000, ABI 7300, ABI 7500, ABI 7900, MX3000P, MX3005P, iCycler iQ4, iCycler iQ5, MJ-chromo4, Light cycler R480, SLAN and other full-automatic fluorescent quantitative PCR instruments.

[0090] The test method is as follows:

[0091] Step one, sample preparation (sample preparation area)

[0092] 1. Eye and nose swab samples, anal swab samples: take a cotton swab into the nose and eyelid back and forth 3-5 times, or take the eye and nose secretions; take another cotton swab into the anus and rotate 2-3 times to take the feces; dip the two cotton swabs with samples into a centrifuge tube with 1.0mL of normal saline (or PBS) for standby.

[0093] 2. Tissue sample: Randomly take a small amount of sample (about 1 g) from three different positions of each tissue, respectively, cut and mix with surgical scissors, grind 0.05 g in a grinder, continue to grind after adding 1.5 mL of normal saline, and transfer the homogenate into a 1.5 mL sterile centrifuge tube after centrifugation at 8000 rpm for 2 min, and take 200 μL of supernatant to a 1.5 mL sterile centrifuge tube for detection.

[0094] 3. Liquid sample: Use a sterile syringe to extract the sample and place it in a sterile centrifuge tube for detection.

[0095] 4. Cross-contamination between samples should be avoided.

[0096] 5. The sample should be detected in time after collection, or can be stored at -20±5°C for detection, and long-term storage should be placed below -70°C.

[0097] 6. Sample nucleic acid extraction: Use nucleic acid extraction or purification reagents, and follow the operation steps according to the instructions of the corresponding extraction reagents. If the extracted DNA or RNA template cannot be detected immediately, it is recommended to be stored below -70°C.

[0098] Step two, reagent preparation (reagent preparation area)

[0099] 1. Take the corresponding reagents of the kit from the -20°C refrigerator 20 min before the experiment, and equilibrate to room temperature (15-25°C), and make the reagents completely melt, and centrifuge for 15 s to make the liquid completely sink to the bottom of the tube.

[0100] 2. Calculate the number of reactions (n) required for the experiment, and calculate the amount of each reagent required for the experiment according to the reaction system preparation method of Table 4 and Table 5. Pay special attention to: K1 and K2 reagents are required for each sample, and the two reagents need to be prepared and packaged separately.

[0101] n = number of negative controls (1 reaction) + number of positive controls (1 reaction) + error allowance + number of samples

[0102] Table 4: K1 hole reaction system preparation method

[0103]

[0104] Table 5: K2 hole reaction system preparation method

[0105]

[0106] Since the pathogen categories detected by K1 and K2 holes are different, the K1 hole reaction system does not need the mixed enzyme liquid component, while the K2 hole reaction system needs the mixed enzyme liquid component.

[0107] 3. Add K1 well and K2 well reagents in appropriate volume of sterile centrifuge tube, mix well and centrifuge at 2000 rpm for 10 s, then dispense the reagents into PCR reaction tubes according to the volume of 20.0 μL / tube, and label K1 and K2.

[0108] 4. Cover the PCR reaction tube and transfer it to the sample preparation area, and store the remaining reagents in the freezer at -20 °C.

[0109] Step three, sample addition:

[0110] 1. Open the PCR reaction tube cover, and add 10 μL of sample template to K1 well and K2 well, respectively, and add 10 μL of positive control and negative control, respectively, and record the sample addition order.

[0111] 2. Cover the PCR tube cover, mix the PCR reaction solution and sample template, and centrifuge at 2000 rpm for 10 s.

[0112] 3. Transfer the PCR reaction tube to the PCR area for machine operation.

[0113] Step four, PCR reaction: (nucleic acid amplification area)

[0114] 1. Start preheating and check the instrument performance.

[0115] 2. Take the PCR reaction tube prepared in the sample preparation area, place it in the corresponding position of the instrument sample slot, and record the placement order.

[0116] 3. Set the instrument nucleic acid amplification related parameters according to Table 6, and perform PCR amplification.

[0117] Table 6: Instrument nucleic acid amplification related parameters

[0118]

[0119] Note: ABI series fluorescence quantitative PCR instrument does not select ROX correction, and the quenching group selects None.

[0120] Step five, result analysis

[0121] 1. Result analysis condition setting:

[0122] Threshold setting principle: adjust the threshold line reasonably, and different instruments can be adjusted according to the instrument noise.

[0123] 2. Kit effectiveness determination:

[0124] 2.1 Positive control: K1 well and K2 well FAM, VI C, Texas Red channel Ct value is ≤30, with obvious exponential growth.

[0125] 2.2 Negative control: Ct value of K1 well and K2 well FAM, VI C, Texas Red channel > 38 or no Ct value, linear is straight line or slight oblique line, no obvious exponential growth.

[0126] 3. Sample result determination:

[0127] 3.1 Positive: sample detection result Ct value < 35, with obvious exponential growth.

[0128] 3.2 Suspicious: sample detection result Ct value is in the range of 35-38, at which time the sample should be repeatedly detected. If the repeated experiment result Ct value is still in the range of 35-38, with obvious exponential growth, it is determined to be positive, otherwise it is negative.

[0129] 3.3 Negative: sample detection result Ct value > 38 or no Ct value, linear is straight line or slight oblique line, no obvious exponential growth.

[0130] 3.4 Sample detection result explanation see Table 7:

[0131]

[0132] Note:

[0133] (1) "+" represents positive detection result, "-" represents negative detection result;

[0134] (2) If the same sample multi-channel detection result is positive, it means that multiple pathogenic nucleic acids are detected in the sample.

[0135] Example 3: Use the above kit to detect nucleic acid samples with known background

[0136] Use the above kit to detect nucleic acid samples with known background (which have been detected by corresponding pathogenic fluorescent PCR detection kit). The results are as follows, which shows that the kit has good specificity and specific amplification of specific sample nucleic acid without non-specific reaction.

[0137]

[0138] Note: Nucleic acid from clinical detection of pathological material or commercial vaccine

[0139] Example 4: Only K1 well in Example 3 is used for detection

[0140] Example 5: Only K2 well in Example 3 is used for detection

[0141]

Notes

[0142] 1. Laboratory management should be strictly in accordance with the relevant national clinical genetic amplification laboratory management specifications. Laboratory personnel must be professionally trained before they can work; the experimental process should be carried out in separate zones (reagent preparation zone, sample preparation zone, amplification and product analysis zone), and special instruments and equipment should be used for each stage of the experimental operation, and the supplies in each zone and stage should not be cross-used; the personnel flow and air flow between zones should have strict requirements.

[0143] 2. Before the experiment, please read the reagent kit manual carefully and strictly follow the operation steps; using components not provided by the reagent kit or not following the manual may lead to incorrect results.

[0144] 3. All reagents should be stored at the specified temperature. The reagents stored at -20℃ should be completely melted before use, centrifuged at 8000 rpm for 15 s to make the liquid sink to the bottom of the tube, placed in an ice box, and the liquid should be taken as much as possible on the surface layer of the liquid when using a pipette. After use, it should be immediately placed back at -20℃.

[0145] 4. The operation table, pipette, centrifuge and other instruments and supplies should be frequently disinfected or soaked with 1.0% sodium hypochlorite or dilute hydrochloric acid, and the consumables should be treated with enzyme removal (it is recommended to soak with 0.1% DEPC water overnight and sterilize). The pipetting and timing processes during the operation must be accurate. The experimental room and clean bench should be treated with ultraviolet light regularly or after each experiment.

[0146] 5. Disposable gloves without fluorescent substances should be used, and they should be replaced frequently to prevent cross-contamination.

[0147] 6. The reagent components in the kit and the waste during the experiment should be treated according to the method for handling potentially infectious substances during use.

[0148] 7. To prevent fluorescence interference, direct contact with hands should be avoided, and please do not mark on the PCR reaction tube.

[0149] 8. Possible reasons for false negative results: improper operation during sample collection, transportation, storage and nucleic acid extraction, which can easily cause nucleic acid degradation and false negative results; sequence changes caused by variation of the target sequence of the virus to be tested or other reasons may lead to false negative results.

[0150] 9. If cross-contamination occurs during sample collection and preparation, false positive results may be easily obtained.

[0151]

Storage conditions and shelf life

[0152] The kit should be stored at -20℃ in the dark;

[0153] The shelf life of the kit is 12 months;

[0154] The number of times of repeated freezing and thawing of the kit should not exceed 5 times.

[0155] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. The RT-qPCR detection primer, fluorescent probe combination for simultaneously detecting three kinds of canine digestive tract pathogens of canine picornavirus, canine Bordetella bronchiseptica and canine adenovirus, characterized in that, The RT-qPCR detection primer, fluorescent probe combination contains: the forward primer CPV-F, the reverse primer CPV-R, the FAM labeled fluorescent probe CPV-P of canine parvovirus VP2 gene; the forward primer Bb-F, the reverse primer Bb-R, the VIC labeled fluorescent probe Bb-P of canine Bordetella FIM2 gene; the forward primer CAV-F, the reverse primer CAV-R, the Texas Red labeled fluorescent probe CAV-P of canine adenovirus S protein gene.

2. The RT-qPCR detection primer and fluorescent probe combination for simultaneously detecting three canine respiratory tract pathogens of canine distemper virus, canine coronavirus and canine parainfluenza virus, characterized in that, The RT-qPCR detection primer, fluorescent probe combination contains: the forward primer CDV-F, the reverse primer CDV-R, the FAM labeled fluorescent probe CDV-P of canine distemper virus N protein gene; the forward primer COV-F, the reverse primer COV-R, the VIC labeled fluorescent probe COV-P of canine coronavirus ORF1 gene; the forward primer CPIV-F, the reverse primer CPIV-R, the Texas Red labeled fluorescent probe CPIV-P of canine parainfluenza virus N protein gene.

3. The RT-qPCR detection primer, fluorescent probe combination of claim 1, wherein, the nucleotide of the CPV-F is shown as sequence SEQ ID NO. 1, the nucleotide of the CPV-P is shown as sequence SEQ ID NO. 2, the nucleotide of the CPV-R is shown as sequence SEQ ID NO. 3; the nucleotide of the Bb-F is shown as sequence SEQ ID NO. 4, the nucleotide of the Bb-P is shown as sequence SEQ ID NO. 5, the nucleotide of the Bb-R is shown as sequence SEQ ID NO. 6; the nucleotide of the CAV-F is shown as sequence SEQ ID NO. 7, the nucleotide of the CAV-P is shown as sequence SEQ ID NO. 8, the nucleotide of the CAV-R is shown as sequence SEQ ID NO.

9.

4. The RT-qPCR detection primer, fluorescent probe combination of claim 2, wherein, the nucleotide of the CDV-F is shown as sequence SEQ ID NO. 10, the nucleotide of the CDV-P is shown as sequence SEQ ID NO. 11, the nucleotide of the CDV-R is shown as sequence SEQ ID NO. 12; the nucleotide of the COV-F is shown as sequence SEQ ID NO. 13, the nucleotide of the COV-P is shown as sequence SEQ ID NO. 14, the nucleotide of the COV-R is shown as sequence SEQ ID NO. 15; the nucleotide of the CPIV-F is shown as sequence SEQ ID NO. 16, the nucleotide of the CPIV-P is shown as sequence SEQ ID NO. 17, the nucleotide of the CPIV-R is shown as sequence SEQ ID NO.

18.

5. The RT-qPCR detection primer and fluorescent probe combination for simultaneously detecting six canine pathogens of canine parvovirus, canine bordetella, canine adenovirus, canine distemper virus, canine coronavirus and canine parainfluenza virus, characterized in that, The RT-qPCR detection primer and fluorescent probe combination comprises two groups of RT-qPCR detection primers and fluorescent probe combinations, which are the RT-qPCR detection primer and fluorescent probe combination in claim 1 and the RT-qPCR detection primer and fluorescent probe combination in claim 2, respectively.

6. A method for detecting nucleic acid of six pathogens of digestive tract and respiratory tract of dogs by fluorescent RT-qPCR, characterized in that, The method comprises the following steps: 1) sample nucleic acid extraction; 2) calculating the number of reactions n required for the experiment = the number of negative controls + the number of positive controls + the number of samples; 3) respectively configuring K1 and K2 two-hole reagents for each reaction, wherein the K1 hole reagent is configured into an RT-qPCR reaction solution according to the RT-qPCR detection primer and fluorescent probe combination in claim 1, and the K2 hole reagent is configured into an RT-qPCR reaction solution according to the RT-qPCR detection primer and fluorescent probe combination in claim 2; adding the configured reagents into corresponding K1 hole and K2 hole PCR reaction tubes, respectively; 4) adding sample nucleic acid templates, positive controls and negative controls into the K1 hole and the K2 hole, respectively, and simultaneously performing RT-qPCR reactions under the same conditions; 5) selecting FAM, VIC and Texas Red three fluorescent channels to collect fluorescence signals and obtain Ct values in the K1 hole and the K2 hole; the K1 hole FAM positive signal can detect canine parvovirus, the K1 hole VIC positive signal can detect canine bordetella, the K1 hole Texas Red positive signal can detect canine adenovirus, the K2 hole FAM positive signal can detect canine distemper virus, the K2 hole VIC positive signal can detect canine coronavirus, and the K2 hole Texas Red positive signal can detect canine parainfluenza virus.

7. The simultaneous detection of canine digestive tract and respiratory tract pathogenic nucleic acid six-union fluorescent RT-PCR detection method according to claim 6, characterized in that, The reaction conditions for simultaneously performing PCR amplification in the K1 hole and the K2 hole are as follows: reverse transcription at 50℃ for 10 min, 1 cycle; pre-denaturation at 95℃ for 30 s, 1 cycle; and PCR at 95℃ for 10 s, 40 cycles.

8. The simultaneous detection of canine digestive tract and respiratory tract pathogenic nucleic acid six-union fluorescent RT-PCR detection method according to claim 6, characterized in that, If the obtained Ct value is ≤ 35 and there is obvious exponential growth, it is determined as a positive signal; if the obtained Ct value is in the range of 35-38, and the Ct value is still in the range of 35-38 after repeated detection of the sample and there is obvious exponential growth, it is determined as a positive signal, otherwise, it is determined as a negative signal; if the obtained Ct value is > 38 or there is no Ct value, the line is a straight line or a slight slant line, and there is no obvious exponential growth, it is determined as a negative signal.

9. A canine gastrointestinal and respiratory pathogen nucleic acid hexaplex fluorescent RT-qPCR detection kit, characterized in that, The kit comprises RT-qPCR reaction solutions, enzyme mixtures, negative quality control products and positive quality control products, wherein the RT-qPCR reaction solutions comprise two groups of RT-qPCR reaction solutions configured respectively, one group of which comprises the RT-qPCR detection primer and fluorescent probe combination in claim 1, and the other group of which comprises the RT-qPCR detection primer and fluorescent probe combination in claim 2.

10. The method of using the Canine Digestive Tract and Respiratory Pathogen Nucleic Acid Six-in-One Fluorescent RT-qPCR Detection Kit according to claim 9, characterized in that, After each group of detection primers and fluorescent probe combinations completes fluorescent RT-qPCR detection of a sample in the same PCR reaction system, Ct values are obtained through FAM, VIC and Texas Red fluorescent detection channels, respectively; and the two groups of RT-qPCR detection primers and fluorescent probe combination reaction systems simultaneously perform RT-qPCR reactions under the same conditions.

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