Primer probe composition for distinguishing babesia canis from babesia gibsoni, detection kit and detection method

Abstract

The invention belongs to the technical field of detection of pathogens of canine babesia, and discloses a primer probe composition, a detection kit and a detection method for distinguishing canine babesia and babesia gibsoni. The primer probe composition comprises an upstream primer gimbal-F of which the sequence is as shown in SEQ ID NO.1, a downstream primer gimbal-R of which the sequence is as shown in SEQ ID NO.2, and a fluorescent probe gimbal-P of which the sequence is as shown in SEQ ID NO.3; the kit comprises an upstream primer Kiba-F with a sequence as shown in SEQ ID NO.4, a downstream primer Kiba-R with a sequence as shown in SEQ ID NO.5, and a fluorescent probe Kiba-P with a sequence as shown in SEQ ID NO.6. The detection kit comprises the primer probe composition. The detection method comprises the steps of sample treatment, fluorescent quantitative amplification and detection result analysis. According to the detection method, the babesia gibsoni and the babesia canis in a sample can be accurately detected at the same time and effectively distinguished, and an accurate medication scheme is provided for dogs infected with the babesia gibsoni.

Description

Technical Field

[0001] The invention relates to the technical field of pathogen detection of canine babesiosis, and in particular to a primer-probe combination, a detection kit and a detection method for distinguishing canine Babesia and Babesia gibsoni. Background Art

[0002] Canine Babesiosis is a disease caused by Babesia parasitizing in dog red blood cells. Babesia is a blood-borne protozoan transmitted by ticks. Dogs infected with Babesia will show symptoms of fever and lethargy, followed by hemolytic anemia, red blood cell rupture, abnormal coagulation of the liver, lungs, and kidneys, electrolyte imbalance, and organ dysfunction. It usually presents an acute course and seriously threatens the health of dogs.

[0003] At present, the occurrence and distribution of canine babesiosis in my country is epidemic and widespread. Spring, summer and autumn are the peak seasons of the year. The disease may be transmitted in any area where ticks can breed, and the incidence rate of the disease has been increasing year by year in recent years.

[0004] The pathogens that cause canine babesiosis in my country are mainly Babesia canis and Babesia gibsoni. Among them, Babesia canis is relatively large, generally 4-5μm long. Babesia canis vogeli, Babesia canis and Babesia canisrossi are the three most common canine Babesia species in my country. Babesia gibsoni is very small, 1-2.5μm long. It is the main pathogenic insect species of babesiosis in my country. Clinically, the clinical symptoms caused by canine Babesia and Babesia gibbsoni are similar, but there are differences in drug treatment. Triazoguanidine and oxydiphenyl ether isethionate are highly effective against canine Babesia but cannot eliminate Babesia gibbsoni. The combination of atovaquone and azithromycin is the most effective treatment for Babesia gibbsoni. Therefore, effective differentiation of pathogenic parasites is of great significance for the prevention and treatment of canine babesiosis.

[0005] Currently, the more direct diagnostic method in clinical practice is blood smear staining after venous blood sampling. This method is not only convenient and quick, but can also distinguish between Babesia gibsoni and Babesia canis based on the size of the worms. However, blood smears are limited by smear and reading techniques, have high requirements for operators, and are difficult to detect worms in the early stages of infection, which can easily lead to missed detections.

[0006] With the development of molecular biology technology, fluorescent quantitative PCR technology has been widely used in the diagnosis of canine babesiosis due to its ultra-high sensitivity and specificity, but there is currently no identification detection method that can effectively distinguish between Babesia gibbsii and Babesia canis. In addition, the existing fluorescent quantitative PCR reagents are cumbersome to prepare, require professional PCR laboratories, and have a long detection cycle of about 2 hours, which limits its promotion and use in pet hospitals.

[0007] Therefore, the prior art needs to be further improved. Summary of the invention

[0008] In view of the above problems, the present invention provides a primer-probe combination, a detection kit and a detection method for quickly and accurately distinguishing Babesia canis from Babesia gibsoni. The detection method is simple to operate, can quickly and effectively distinguish Babesia gibsoni from Babesia canis in a sample, is suitable for promotion and application, and is of great significance for the prevention and treatment of canine babesiosis.

[0009] In a first aspect, the present application provides a primer-probe composition for distinguishing Babesia canis from Babesia gisoni, comprising: Primer probes for detecting the specific gene TRAP of Babesia gibsoni: upstream primer Giba-F having a sequence as shown in SEQ ID NO.1, downstream primer Giba-R having a sequence as shown in SEQ ID NO.2, and fluorescent probe Giba-P having a sequence as shown in SEQ ID NO.3; Primer probe for detecting 16s rRNA of Babesia canis: upstream primer Canis-F with a sequence as shown in SEQ ID NO.4, downstream primer Canis-R with a sequence as shown in SEQ ID NO.5, and fluorescent probe Canis-P with a sequence as shown in SEQ ID NO.6; The 5' end of the fluorescent probe is labeled with a fluorescent reporter group, and the 3' end of the fluorescent probe is labeled with a fluorescent quencher group.

[0010] Preferably, the primer-probe composition further comprises primer probes for detecting the internal reference gene ACTB: an upstream primer ACTB-F having a sequence as shown in SEQ ID NO.7, a downstream primer ACTB-R having a sequence as shown in SEQ ID NO.8, and a fluorescent probe ACTB-P having a sequence as shown in SEQ ID NO.9.

[0011] Optionally, in the primer-probe composition, the fluorescent reporter group is selected from FAM, HEX, CY5, ROX, and VIC; and the fluorescent quencher group is selected from BHQ1, BHQ2, and BHQ3.

[0012] In a second aspect, the present application also provides a detection kit for distinguishing Babesia canis from Babesia gibsoni, which comprises the aforementioned primer-probe combination.

[0013] Optionally, the detection kit includes a freeze-dried PCR reagent, which is prepared by freeze-drying a PCR mixture, wherein the PCR mixture includes: the aforementioned primer-probe combination, DNA polymerase, dNTPs, a buffer and a freeze-drying protective agent; Preferably, the lyoprotectant consists of the following ingredients: 4% W / V% trehalose and 3% W / V% dextran; Preferably, the PCR mixture is mainly prepared from the following ingredients: 0.1-1 μM primer probe, 0.2-0.8 μl super Taq DNA polymerase, 2.5 μl 10x buffer, and 12.5 μl lyophilized protective agent.

[0014] Preferably, the concentration of the primers in the freeze-dried PCR reagent in the kit is 0.4-1 μM; the concentration of the probe is 0.1-0.5 μM.

[0015] Preferably, the DNA polymerase is super Taq DNA polymerase. The super Taq DNA polymerase is a modified and optimized Taq DNA polymerase, which has better tolerance to inhibitors and higher amplification and extension efficiency; Optionally, in the detection kit, the PCR mixture further includes: a PCR denaturation accelerator, which is a combination of one or more of the following ingredients: 1%-15% W / V% betaine, 1%-15% V / V% DMSO and 1%-15% V / V% formamide.

[0016] Preferably, the PCR mixture further comprises: a nucleic acid releaser; the nucleic acid releaser comprises the following components in the following concentrations: 100 mM KOH, 5 mM EDTA, 100 mM Tris-HCl and 5% V / V% sorbitol; Optionally, the PCR mixture also includes a rehydration solution, which is a sterile aqueous solution containing 5% V / V% glycerol.

[0017] Preferably, the PCR denaturation accelerator is 1-15% V / V% DMSO. More preferably, the PCR denaturation accelerator is 15% V / V% DMSO.

[0018] Optionally, the detection kit further includes: a positive quality control and a negative quality control, the positive quality control is a mixture of Babesia gibbsii plasmid, Babesia canis plasmid and canine ACTB internal reference plasmid, the ratio of the three being 1:1:1; the negative control is sterile deionized water.

[0019] In a third aspect, a method for preparing the aforementioned freeze-dried PCR reagent comprises the following steps: after mixing all the components, freeze-drying them through a multi-stage freeze-drying procedure, wherein the multi-stage freeze-drying procedure is: Pre-freezing stage: -45℃, maintain for 2h; primary sublimation drying: heat to -30℃, turn on the vacuum pump and quickly evacuate to 1mbar, maintain for 2h; then heat to -15℃, maintain for 2h; secondary sublimation drying: continue to heat to 10℃, maintain for 2h; after the end, heat to 25℃, maintain for 2h. Capping: after the freeze-drying program is completed, nitrogen is flushed in to balance to atmospheric pressure, and then eight rows of tube caps are capped and sealed to complete freeze drying.

[0020] In a fourth aspect, the present application also provides a detection method for distinguishing Babesia canis from Babesia gibsoni for non-disease diagnosis purposes, which comprises the following steps: S1. Add a nucleic acid release agent to the sample, vortex and shake, pipette the suspension and add it to the reconstituted solution, shake and mix, add the mixture to the freeze-dried PCR reagent, mix and centrifuge to obtain a reaction mixture; S2. The reaction mixture is subjected to ultra-fast fluorescence quantitative PCR amplification; the ultra-fast fluorescence quantitative PCR amplification procedure is: The denaturation temperature is 88-95°C and the reaction time is 1-8s; The annealing temperature is 58-70°C, the reaction time is 3-15s, and fluorescence is collected; The number of reaction cycles was 35 to 45 cycles; S3. Analyze the Ct values ​​of the three channels in the fluorescent quantitative PCR reaction system to determine whether the sample is infected with Babesia gibsoni and / or Babesia canis.

[0021] The above detection method for distinguishing Babesia canis from Babesia gibsoni is simple to operate, has high specificity and accuracy, and greatly shortens the detection cycle. The total time from adding samples to issuing reports can be as short as 20-30 minutes, which has the advantages of short cycle and high efficiency.

[0022] Optionally, the S2 step is performed on an ultra-fast fluorescence quantitative PCR instrument.

[0023] Preferably, the ultra-fast fluorescent quantitative PCR instrument is small in size and light in weight, and comprises 8 PCR reaction wells and 4 fluorescent channels, wherein the 4 fluorescent channels are FAM, HEX, ROX and CY5.

[0024] Further preferably, in step S2, the ultra-fast fluorescent quantitative PCR amplification program is: 90° C., 2 s; 65° C., 5 s; amplification is performed for 40 cycles.

[0025] Preferably, in step S3, intelligent result analysis report interpretation software is used, which contains various pre-stored common project databases and can generate interpretation reports by automatically identifying fluorescent quantitative PCR results. In practical applications, it is more convenient to quickly display experimental results.

[0026] Optionally, in the detection method, the specific analysis and judgment method of step S3 is: If the fluorescence channel of the fluorescent probe Jiba-P is the first fluorescence channel, the fluorescence channel used by the fluorescent probe Dogba-P is the second fluorescence channel; When the Ct value of the first fluorescent channel is <40 and there is no Ct value in the second fluorescent channel, it means that the sample contains only Babesia gibsoni; When there is no Ct value in the first fluorescence channel and the Ct value in the second fluorescence channel is <40, it means that only Babesia canis is present in the sample; When the Ct value of the first fluorescence channel is <40 and the Ct value of the second fluorescence channel is <40, it indicates that the sample contains Babesia gibsoni and Babesia canis; When there is no Ct value in the first fluorescent channel and no Ct value in the second fluorescent channel, it means that there is no Babesia gibbsii and Babesia canis in the sample.

[0027] Optionally, in the detection method, if the fluorescent channel used by the fluorescent probe ACTB-P is the third fluorescent channel, when the Ct value of the third fluorescent channel is <40, it indicates that the sample sampling is qualified.

[0028] Further preferably, the first fluorescent channel is FAM, the second fluorescent channel is HEX, and the third fluorescent channel is CY5.

[0029] The present invention has the following beneficial effects: 1. The present invention realizes accurate detection of Babesia gibsoni and Babesia canis in the same PCR reaction system for the first time, and effectively distinguishes Babesia gibsoni and Babesia canis according to their specific gene sequences, providing an accurate medication regimen for dogs infected with Babesia, which is of great significance for the prevention and treatment of canine babesiosis and has a great promoting effect on the healthy development of my country's dog breeding industry.

[0030] 2. The present invention adopts direct release of blood samples, fully premixed freeze-dried PCR reagents, ultra-fast nucleic acid amplification program and a matching ultra-fast fluorescent quantitative PCR detection and analysis system. The equipment is small and convenient, easy to operate, the reagent kit can be transported and stored at room temperature, the detection time is short, and the report interpretation is available immediately. From adding the sample to issuing the report, it takes ≤30 minutes, which is conducive to promotion and use in pet hospitals.

[0031] 3. The present invention uses the PCR-fluorescent probe method, and the amplification reaction and detection adopt a completely closed system to avoid false positives caused by aerosol pollution. The operation method is simple, the detection cycle is short, the detection cost is low, and the sensitivity and specificity are good. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The amplification curves of the three sets of primers and probes of Example 2, Comparative Example 1 and Comparative Example 2 respectively amplify the positive plasmid; Figure 2 The amplification curve results of the three sets of primers and probes of Example 2, Comparative Example 1 and Comparative Example 2 respectively amplified the samples of Babesia gisoni; wherein Figure 2 A is the result of amplifying Babesia gibsoni samples using the canine primers among the three sets of primer probes; Figure 2 B is the result of amplifying Babesia gibsoni samples using the Giba primers among the three sets of primer probes; Figure 3 The amplification curve results of the three sets of primers and probes of Example 2, Comparative Example 1 and Comparative Example 2 respectively amplified the canine Babesia samples; wherein Figure 3 A is the result of amplifying Babesia canis samples using the canine Babesia primers among the three sets of primer probes; Figure 3 B is the result of amplifying the canine Babesia sample using the Giba primers among the three sets of primer probes; Figure 4 The kit for detecting canine Babesia canis 1x10 1 ~1x10 6 The fluorescence quantitative amplification results of the gradient dilution plasmid with 10 copies / ul; Figure 5 The kit of Example 4 detects Babesia gibbsii 1x10 1 ~1x10 6 The results of fluorescence quantitative amplification of the gradient dilution plasmid of copies / ul; Figure 6 The kit of Example 4 detects the canine reference gene ACTB 1x10 1 ~1x10 6 The fluorescence quantitative amplification results of the gradient dilution plasmid of copies / ul. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. These embodiments are only used to illustrate the detection method and process described in the present invention, and are not used to limit the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0034] Example 1 Screening of primer-probe combination and setting of kit 1. Design of primer-probe combination 40 TRAP gene sequences of Babesia gibsoni were obtained from NCBI, and the sequences were aligned using Clustal X2 software to find out the relatively conservative gene sequences, and several pairs of upstream and downstream primers and probes for specific amplification of Babesia gibsoni were designed.

[0035] 40 16s rRNA sequences of Babesia canis and 20 16s rRNA sequences of Babesia gibsoni were obtained from NCBI. Sequences were aligned using Clustal X2 software to identify multiple gene sequences that were relatively conservative for Babesia canis but specific for Babesia gibsoni. Multiple pairs of upstream and downstream primers and probes were designed accordingly.

[0036] The canine ACTB gene sequence was obtained from NCBI, and the gene was used as an internal reference gene, and upstream and downstream primers and probes were designed. The primers of the internal reference gene were used to avoid false negative interference with the test results. In other embodiments, other conserved genes of the canine can be selected as internal reference genes.

[0037] 2. Screening and Validation of Primer-probe Combinations Through multiple samples and plasmids, primer probe combinations were verified and screened, and primer probe combinations with good specificity, high sensitivity, and high amplification efficiency for simultaneously distinguishing Babesia canis and Babesia gibbsii were screened out. The specific primer probe information is shown in Table 1: Table 1 Primer and probe sequences of Babesia gibsoni, Babesia canis and ACTB genes

[0038] 3. Preparation of the Kit This embodiment designs a kit for use with respect to the multiple sets of primers and probes in Table 1 above.

[0039] As shown in Table 2, the kit includes: freeze-dried PCR reagents, nucleic acid release agent, reconstitution solution, positive quality control and negative control.

[0040] Among them, the freeze-dried PCR reagent includes the following components: 0.5μM Jiba-F, 0.5μM Jiba-R, 0.25μM Jiba-P, 0.5μM Dogba-F, 0.5μM Dogba-R, 0.25μM Dogba-P, 0.4μMACTB–F, 0.4μM ACTB–R, 0.2μM ACTB–P; 0.6ul super Taq DNA polymerase, 2.5μl 10x buffer, PCR denaturation accelerator 15% v / v% DMSO and 12.5ul freeze-drying protective agent, and it is divided into PCR eight-tube strips and then freeze-dried according to a multi-stage freeze-drying program.

[0041] The freeze-drying procedure is as follows: pre-freezing: -45°C, maintain for 2 hours; primary sublimation drying: heating to -30°C, while turning on the vacuum pump to quickly evacuate to 1mbar, maintain for 2 hours; then heating to -15°C, maintain for 2 hours; secondary sublimation drying: continue heating to 10°C, maintain for 2 hours; after the end, heating to 25°C, maintain for 2 hours. Capping: After the freeze-drying program is completed, nitrogen is flushed in to balance to atmospheric pressure, and then eight rows of tube caps are capped and sealed to complete the freeze-drying.

[0042] The concentration of primers in the freeze-dried PCR reagent is 0.4-1 μM; the concentration of probes is 0.1-0.5 μM.

[0043] The nucleic acid releaser is used for releasing nucleic acid from a blood sample, and contains components with the following concentrations: 100 mM KOH, 5 mM EDTA, 100 mM Tris-HCl, and 5% v / v% sorbitol.

[0044] The reconstitution solution is used to reconstitute the entire premixed lyophilized reagent, which contains 5% v / v% glycerol in sterile deionized water.

[0045] The positive quality control is a mixed solution prepared from a Babesia gissimii plasmid, a Babesia canis plasmid and a canine ACTB internal reference plasmid at a molar ratio of 1:1:1. The target gene sequence of the Babesia gissimii plasmid is shown in SEQ ID NO.10 in the sequence list, the target gene of the Babesia canis plasmid is shown in SEQ ID NO.11 in the sequence list, and the target gene sequence of the ACTB plasmid is shown in SEQ ID NO.12 in the sequence list.

[0046] The lyoprotectant consists of the following ingredients: 4% W / V% trehalose and 3% W / V% dextran.

[0047] Table 2 Composition of the kit

[0048] 4. This embodiment also provides a detection method for distinguishing Babesia canis from Babesia gibsoni using the above method, the detection method comprising the following steps: S1. Draw 1 ml of canine venous blood using an EDTA blood collection tube; and take out various reagents in the kit; Add the nucleic acid release agent to 25ul of the sample to be tested (i.e. the above-mentioned canine venous blood), vortex and shake for 2 minutes, take out 25ul of the mixed solution and add it to the re-solution, vortex and shake to mix, take out 25ul and add it to the lyophilized reagent.

[0049] At the same time, take 25ul of positive quality control and negative control and add them to the corresponding lyophilized reagents as positive and negative controls. Cover the tubes, mix well and centrifuge.

[0050] S2, placing the centrifuged reaction mixture into a rapid fluorescence quantitative PCR detection and analysis system (i.e., an ultra-rapid fluorescence quantitative PCR instrument), and performing ultra-rapid fluorescence quantitative PCR amplification according to the following amplification procedure; The ultra-fast fluorescence quantitative PCR amplification program was: 90°C, 2s; 65°C, 5s (collecting fluorescence); and amplification was performed for 40 cycles.

[0051] S3. Analyze the Ct values ​​of the three channels in the fluorescent quantitative PCR reaction system to determine whether the sample is infected with Babesia gibsoni and / or Babesia canis.

[0052] The specific analysis and judgment methods are as follows: (as shown in Table 3).

[0053] When the Ct value of the FAM channel is <40 and there is no Ct value in the HEX channel, it means that the sample contains only Babesia gibsoni; When there is no Ct value in the FAM channel and the Ct value in the HEX channel is <40, it means that only Babesia canis is present in the sample; When the Ct value of the FAM channel is <40 and the Ct value of the HEX channel is <40, it indicates that the sample contains Babesia gibsoni and Babesia canis; When there is no Ct value in the FAM channel and no Ct value in the HEX channel, it means that there is no Babesia gibbsii and Babesia canis in the sample; When the Ct value of the CY5 channel is <40, it means that the sample sampling is qualified.

[0054] In other embodiments, the above three fluorescent channels may be replaced by other types of fluorescent channels, and the solution of the present invention is not limited to the specific configuration of this embodiment.

[0055] Table 3 Analysis and judgment methods

[0056] Note: If there is no Ct value in the CY5 channel, retesting is required.

[0057] Example 2 Comparison of the effects of primer-probe combinations 1. Experimental methods This experiment provides kits of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4. The difference between these four kits and the kit of Example 1 is that different primer probe compositions are used, as shown in Table 1. The fluorescent probes on each primer probe of each control group refer to the settings of Example 1 for comparative analysis, and the preparation method of the kit of each group is the same as that of the aforementioned Example 1.

[0058] Table 4 Primer probes for the control group

[0059] The kits prepared in Example 1 of the present application, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 were respectively subjected to the following experiment on the same sample according to the detection method of Example 1, and the common amplification procedure of 95° C., 5 min; (95° C., 15 s, 60° C., 30 s (collecting fluorescence), 40 cycles): (1) The Babesia gibbsii-specific primers and Babesia canis-specific primers of Example 1, Control Group 1, Control Group 2, Control Group 3 and Control Group 4 were used to amplify the Babesia gibbsii-positive plasmid and the Babesia canis-positive plasmid in the kit of Example 1, respectively, and the amplification effect of each primer pair was analyzed.

[0060] (2) The Babesia gibbsii-specific primers and Babesia canis-specific primers of Example 1, Control Group 1, Control Group 2, Control Group 3 and Control Group 4 were used to amplify Babesia gibbsii samples and Babesia canis samples, respectively, and the amplification effect of each primer pair was analyzed.

[0061] Compare the experimental results of each kit.

[0062] 2. Experimental results and analysis like Figure 1 As shown, from the results of amplifying the positive plasmid with the above five sets of primers, it can be seen that in the multiple reaction system, the amplification differences among the five sets of primer probes of Jiba and Dogba are not much, and the corresponding plasmids can be effectively amplified. However, the amplification of the internal reference ACTB in Comparative Examples 3 and 4 is significantly inhibited, and the CT value is significantly delayed (the value is increased), and the amplification of the internal reference ACTB in Comparative Examples 1 and 2 is slightly inhibited.

[0063] Figure 2 The results of five sets of primers amplifying Babesia gibsoni samples are shown in Figure 2. Figure 2As shown in A, the results of amplifying the Babesia gibbsii samples using the five sets of primers of the dog bar are that the dog bar primers of Comparative Examples 1, 3 and 4 have non-specific amplification of the Babesia gibbsii samples, and the dog bar primers of Comparative Example 2 and Example 1 have no non-specific amplification of the Babesia gibbsii. Figure 2 As shown in B, the results of amplifying the Babesia gibbsii samples using the five sets of primers of Jiba showed that all five sets of primers could amplify Babesia gibbsii, and the Ct values ​​of Comparative Examples 1, 3 and 4 were earlier than those of Comparative Example 2 and Example 1.

[0064] Figure 3 The results of five sets of primers amplifying canine Babesia samples are shown in Figure 2. Figure 3 As shown in A, all five sets of primers can amplify the dog sample, among which the amplification Ct values ​​of Comparative Examples 1, 3 and 4 are advanced, and Comparative Example 2 is the same as Example 1. Figure 3 As shown in B, the five sets of primers of Giba were used to amplify the canine Babesia sample. Comparative Example 1 had non-specific amplification of the canine Babesia sample, while Comparative Examples 2, 3, 4 and Example 1 had no non-specific amplification.

[0065] From the above analysis, it can be seen that the primer-probe compositions of the kits of Comparative Examples 1, 3 and 4 cannot effectively distinguish Babesia gisoni from Babesia canis, and Comparative Examples 3 and 4 severely inhibit the amplification of the internal reference ACTB. The primer-probe compositions of the kits of Examples 1 and 2 can effectively distinguish Babesia gisoni from Babesia canis, and therefore, Examples 1 and 2 are preferred.

[0066] Example 3 Optimization of the amplification procedure in the detection method of the present application 1. Test methods The kits of Example 1 and Comparative Example 2 were used, and the following different amplification procedures were followed. The setting of the amplification system was referenced to Example 1, and a mixed sample of Canis familiaris and Gibbon positive was used for comparative verification.

[0067] Program 1: Common fluorescence PCR amplification program, pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s (collecting fluorescence), 40 cycles; Program 2: denaturation at 95°C for 2 seconds, annealing at 60°C for 5 seconds (fluorescence collection), 40 cycles; Program 3: denaturation at 90°C for 2 s, annealing at 60°C for 5 s (fluorescence collection), 40 cycles; Program 4: denaturation at 90°C for 2 s, annealing at 65°C for 5 s (fluorescence collection), 40 cycles; 2. Experimental results and analysis: As shown in the results of Table 5: there is little difference in the amplification results between Program 1 and Program 2. Program 1 is a commonly used ordinary PCR amplification program. Program 2 removes the pre-denaturation step based on Program 1, which has little effect on the kit; Program 3 lowers the denaturation temperature to 90°C, and Example 1 and Comparative Example 2 can still effectively amplify positive samples, and there is no significant difference from Program 1; Program 4 lowers the denaturation temperature to 90°C and increases the annealing temperature to 65°C. The sample of Example 1 can still be amplified and there is no significant difference from Program 1, but Comparative Example 2 cannot be amplified. Therefore, the primer-probe combination of Comparative Example 2 is not as good as the primer-probe combination of Example 1 of the present application, and the kit of Example 1 of the present application is better.

[0068] In addition, the results in Table 5 also show that the scheme of Example 1 of the present application can be effectively amplified in procedures 2 to 4, and the amplification effect is not much different from that of conventional procedure 1, but its amplification cycle is shorter and the efficiency is significantly improved.

[0069] Table 5 Comparison of amplification by different programs

[0070] Example 4: Screening and Optimization of Denaturation Accelerators in Example 1 of the Present Application 1. Experimental methods: (1) Reagent preparation: A: Prepare reagent A according to 0.5μM Giba-F, 0.5μM Giba-R, 0.25μM Giba-P, 0.5μM Dogba-F, 0.5μM Dogba-R, 0.25μM Dogba-P, 0.5μM ACTB–F, 0.5μM ACTB–R, 0.25μM ACTB–P; 0.6ul Taq DNA polymerase, 2.5ul 10x buffer, 1ul dNTPs, 15% V / V% DMSO.

[0071] B: Prepare reagent B according to 0.5μM Giba-F, 0.5μM Giba-R, 0.25μM Giba-P, 0.5μM Dogba-F, 0.5μM Dogba-R, 0.25μM Dogba-P, 0.5μM ACTB–F, 0.5μM ACTB–R, 0.25μM ACTB–P; 0.6ul Taq DNA polymerase, 2.5ul 10x buffer, 1ul dNTPs, 10% V / V% DMSO.

[0072] C: Prepare reagent C according to 0.5μM Giba-F, 0.5μM Giba-R, 0.25μM Giba-P, 0.5μM Dogba-F, 0.5μM Dogba-R, 0.25μM Dogba-P, 0.5μM ACTB–F, 0.5μM ACTB–R, 0.25μM ACTB–P; 0.6ul Taq DNA polymerase, 2.5ul 10x buffer, 1ul dNTPs, 5% V / V% DMSO.

[0073] D: Prepare reagent D according to 0.5μM Giba-F, 0.5μM Giba-R, 0.25μM Giba-P, 0.5μM Dogba-F, 0.5μM Dogba-R, 0.25μM Dogba-P, 0.5μM ACTB–F, 0.5μM ACTB–R, 0.25μM ACTB–P; 0.6ul Taq DNA polymerase, 2.5ul 10x buffer, 1ul dNTPs, 15% V / V% formamide.

[0074] E: Prepare reagent E according to 0.5μM Giba-F, 0.5μM Giba-R, 0.25μM Giba-P, 0.5μM Dogba-F, 0.5μM Dogba-R, 0.25μM Dogba-P, 0.5μM ACTB–F, 0.5μM ACTB–R, 0.25μM ACTB–P; 0.6ul Taq DNA polymerase, 2.5ul 10x buffer, 1ul dNTPs, 15% W / V% betaine.

[0075] The ultra-fast fluorescence quantitative PCR amplification program was: 90°C, 2s; 65°C, 5s (collecting fluorescence); and amplification was performed for 40 cycles.

[0076] (2) Loading samples onto the test machine: Follow the operating method of Example 1 and use 2 known positive blood samples for comparative verification.

[0077] 2. Experimental results and analysis: As shown in the experimental results in Table 6, compared with reagent A, the Ct value of the sample amplification of reagent B is significantly delayed, affecting the detection limit (i.e., sensitivity) of the product, the sample amplification of reagent C is not amplified, and the Ct values ​​of the sample amplification of reagent D and reagent E are slightly delayed. Therefore, reagent A is preferred.

[0078] Table 6 Optimization of different denaturants

[0079] Example 5 Performance analysis experiment of the kit of Example 1 The kit of Example 1 was operated according to the detection method of Example 1, and the following performance tests in terms of sensitivity, precision, accuracy, specificity, anti-interference, etc. were performed.

[0080] (1) Sensitivity test ① Experimental methods: The synthetic plasmid standard of Babesia canis, the synthetic plasmid standard of Babesia gibbsii and the synthetic plasmid standard of canine internal reference gene ACTB were diluted in series, and the dilution concentration was 1x10 1 ~1x10 6 Each dilution gradient was tested and analyzed using the kit of Example 1 of the present invention, and each minimum detection limit standard was tested 20 times to evaluate the sensitivity of the kit, and a standard curve was made to evaluate the linearity of the kit.

[0081] ② Experimental results and analysis A. The experimental results show that the kit has a high sensitivity to Babesia canis, which can reach 1x10 1 copies / ul. Figure 4 As shown in the results, the above kit was used to detect 1x10 canine Babesia 1 ~1x10 6 The minimum detection limit of the plasmid was 1x10 copies / ul. 1 copies / ul, and each minimum detection limit standard was tested 20 times, with a positive detection rate of 100%. The verification results are shown in Table 7.

[0082] B. Figure 5 The above kit was used to detect Babesia gibbsii 1x10 1 ~1x10 6 The fluorescence quantitative amplification results of the gradient dilution plasmid of copies / ul show that it has high sensitivity for the detection of Babesia gibbsii and can detect 1x10 1 copies / ul.

[0083] C. Figure 6 The above kit is used to detect the canine reference gene ACTB 1x10 1 ~1x10 6 The fluorescence quantitative amplification results of the gradient dilution plasmid of copies / ul show that the kit has high sensitivity for the detection of canine internal reference gene ACTB and can detect 1x10 1 copies / ul.

[0084] Table 7 Sensitivity test results

[0085] (2) Precision (repeatability) ① Experimental methods: Dilute the Babesia gibbsii plasmid standard and the Babesia canis plasmid standard to 1x10 4 copies / ul and 1x10 2 The test was repeated 10 times, and the coefficient of variation and standard deviation were calculated to evaluate the repeatability.

[0086] ②Experimental results: As shown in Table 8, the standard deviations of the Ct values ​​of different concentrations of Babesia gissimii were 0.21 and 0.30, and the coefficients of variation were 0.80% and 0.89%, respectively; the standard deviations of the Ct values ​​of different concentrations of Babesia canis were 0.03 and 0.25, and the coefficients of variation were 0.13% and 0.75%, respectively; the standard deviations of the Ct values ​​of different concentrations of canine internal reference ACTB were 0.16 and 0.38, and the coefficients of variation were 0.58% and 1.12%, respectively. The above results show that the kit has good repeatability in the detection of Babesia gissimii, Babesia canis, and canine internal reference ACTB.

[0087] Table 8 Precision verification results of Babesia gibsoni, Babesia canis and canine internal reference ACTB

[0088] (3) Accuracy ① Experimental methods The above kit was used to detect clinical Babesia gibbsii samples G1, G2, G3, G4 and G5, and canine Babesia samples C1, C2, C3, C4 and C5, which were confirmed positive by sequencing, to evaluate the accuracy of the kit.

[0089] ②Experimental results: The verification results are shown in Table 9. From the results, it can be seen that the positive detection rate of the kit is 100%, and the negative detection rate is 100%, which shows that the detection accuracy of the kit is high.

[0090] Table 9 Accuracy test results of the kit

[0091] (4) Specificity ① Experimental methods The above kit was used to detect negative reference samples N1-N8 (samples were determined to be negative for Babesia canis and Babesia gibbsii by sequencing, and positive for other pathogens) to evaluate the analytical specificity of the kit. The detection method is referred to Example 1. Among them, the negative reference sample N1 is Leptospira, N2 is Toxoplasma, N3 is Haemophilus, N4 is Hepatozoon, N5 is Anaplasma phagocytophilum, N6 is Ehrlichia canis, N7 is Borrelia burgdorferi, and N8 is Heartworm.

[0092] ②Experimental results: The experimental results showed that the test results of negative reference samples N1~N8 were all negative, with no crossover. This result shows that the detection specificity of the kit is strong.

[0093] (5) Anti-interference ① Experimental methods The kit of the present invention was used to detect common interfering substances in blood that have an inhibitory effect on PCR, including: 60g / L albumin, 37mmol / L triglyceride, 342umol / L bilirubin, and 200g / L hemoglobin, and the interfering substances were added to the positive samples for detection. The anti-interference ability of the kit was evaluated.

[0094] ②Experimental results: The experimental results are shown in Table 10. The common interfering substances in the blood (albumin, triglycerides, bilirubin and hemoglobin) mentioned above have no interference with the test results. This result shows that the test kit has strong anti-interference ability.

[0095] Table 10

[0096] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A primer-probe combination for distinguishing Babesia canis from Babesia gibsoni, characterized in that: include: Primer probes for detecting the specific gene TRAP of Babesia gibsoni: upstream primer Giba-F having a sequence as shown in SEQ ID NO.1, downstream primer Giba-R having a sequence as shown in SEQ ID NO.2, and fluorescent probe Giba-P having a sequence as shown in SEQ ID NO.3; Primer probe for detecting 16s rRNA of Babesia canis: upstream primer Canis-F with a sequence as shown in SEQ ID NO.4, downstream primer Canis-R with a sequence as shown in SEQ ID NO.5, and fluorescent probe Canis-P with a sequence as shown in SEQ ID NO.6; The 5' end of the fluorescent probe is labeled with a fluorescent reporter group, and the 3' end of the fluorescent probe is labeled with a fluorescent quencher group.

2. The primer-probe combination for distinguishing Babesia canis from Babesia gibsoni according to claim 1, characterized in that: It also includes primer probes for detecting the internal reference gene ACTB: an upstream primer ACTB-F with a sequence as shown in SEQ ID NO.7, a downstream primer ACTB-R with a sequence as shown in SEQ ID NO.8, and a fluorescent probe ACTB-P with a sequence as shown in SEQ ID NO.

9.

3. The primer-probe combination for distinguishing Babesia canis from Babesia gissimii according to claim 1, characterized in that: The fluorescent reporter group is selected from FAM, HEX, CY5, ROX, and VIC; the fluorescent quencher group is selected from BHQ1, BHQ2, and BHQ3.

4. A detection kit for distinguishing Babesia canis from Babesia gisoni, characterized in that: Comprising the primer-probe combination as described in any one of claims 1 to 3.

5. The detection kit for distinguishing Babesia canis from Babesia gibsoni according to claim 4, characterized in that: The invention comprises a freeze-dried PCR reagent, wherein the freeze-dried PCR reagent is prepared by freeze-drying a PCR mixture, wherein the PCR mixture comprises: the primer-probe combination according to claim 1, a DNA polymerase, dNTPs, a buffer and a freeze-drying protective agent; Preferably, the PCR mixture is mainly prepared from the following ingredients: 0.1-1 μM primer probe, 0.2-0.8 μl super Taq DNA polymerase, 2.5 μl 10x buffer, and 12.5 μl lyophilized protective agent.

6. The detection kit for distinguishing Babesia canis from Babesia gisoni according to claim 5, characterized in that: The PCR mixture further comprises: a PCR denaturation accelerator, wherein the PCR denaturation accelerator is a combination of one or more of the following components: 1%-15% W / V% betaine, 1%-15% V / V% DMSO and 1%-15% V / V% formamide; Preferably, the PCR mixture further comprises: a nucleic acid releaser; the nucleic acid releaser comprises components in the following concentrations: 100 mM KOH, 5 mM EDTA, 100 mM Tris-HCl and 5% W / V% sorbitol.

7. The detection kit for distinguishing Babesia canis from Babesia gissimii according to claim 5, characterized in that: Also includes: Positive quality control and negative quality control, the positive quality control is a mixture of Babesia gibbsii plasmid, Babesia canis plasmid and canine ACTB internal reference plasmid, the ratio of the three is 1:1:1; the negative control is sterile deionized water.

8. A method for preparing a freeze-dried PCR reagent as claimed in claim 5, characterized in that: The method comprises the following steps: after mixing all the ingredients, freeze-drying is performed through a multi-stage freeze-drying process, wherein the multi-stage freeze-drying process is: Pre-freezing stage: -45℃, maintain for 2h; primary sublimation drying: heat to -30℃, turn on the vacuum pump and quickly evacuate to 1mbar, maintain for 2h; then heat to -15℃, maintain for 2h; secondary sublimation drying: continue to heat to 10℃, maintain for 2h; after the end, heat to 25℃, maintain for 2h. Capping: after the freeze-drying program is completed, nitrogen is flushed in to balance to atmospheric pressure, and then eight rows of tube caps are capped and sealed to complete freeze drying.

9. A detection method for distinguishing Babesia canis from Babesia gibsoni for non-disease diagnosis purposes, characterized in that: Using the detection kit as claimed in claim 4, comprising the following steps: S1. Sample processing: adding the sample to the nucleic acid release agent, vortexing, pipetting the suspension and adding it to the reconstitution solution, vortexing and mixing, adding the mixture to the freeze-dried PCR reagent, mixing and centrifuging to obtain a reaction mixture; S2. Fluorescence quantitative amplification: The above reaction mixture is subjected to ultra-fast fluorescence quantitative PCR amplification; the ultra-fast fluorescence quantitative PCR amplification procedure is: The denaturation temperature is 88-95°C and the reaction time is 1-8s; The annealing temperature is 58-70°C, the reaction time is 3-15s, and fluorescence is collected; The number of reaction cycles was 35 to 45 cycles; S3. Analysis and judgment of test results: According to the Ct values ​​of the three channels in the fluorescence quantitative PCR reaction system, it is judged whether the sample to be tested is infected with Babesia gibsoni and / or Babesia canis.

10. The detection method according to claim 9, characterized in that: The specific analysis and judgment method of step S3 is: If the fluorescence channel of the fluorescent probe Jiba-P is the first fluorescence channel, the fluorescence channel used by the fluorescent probe Dogba-P is the second fluorescence channel; When the Ct value of the first fluorescent channel is <40 and there is no Ct value in the second fluorescent channel, it means that the sample contains only Babesia gibsoni; When there is no Ct value in the first fluorescence channel and the Ct value in the second fluorescence channel is <40, it means that only Babesia canis is present in the sample; When the Ct value of the first fluorescence channel is <40 and the Ct value of the second fluorescence channel is <40, it indicates that the sample contains Babesia gibsoni and Babesia canis; When there is no Ct value in the first fluorescent channel and no Ct value in the second fluorescent channel, it means that there is no Babesia gibbsii and Babesia canis in the sample.

Images