Detection method for distinguishing babesia canis from babesia gibsoni without disease diagnosis purpose

Abstract

The invention belongs to the technical field of canine babesia pathogen detection, and discloses a detection method for distinguishing canine babesia and babesia gibsoni, the detection method comprises the following steps: S1, sample treatment: adding a sample into a nucleic acid releasing agent, carrying out vortex oscillation, sucking suspension, adding the suspension into a reconstitution fluid, oscillating, uniformly mixing, adding a freeze-dried PCR reagent, and carrying out freeze-drying on the freeze-dried PCR reagent; centrifuging to obtain a reaction mixture; s2, fluorescent quantitative amplification: carrying out ultra-fast fluorescent quantitative PCR amplification according to the following procedures: the denaturation temperature is 88-95 DEG C, and the reaction time is 1-8 seconds; the annealing temperature is 58-70 DEG C, the reaction time is 3-15 seconds, and collecting fluorescence; the reaction cycle number is 35-45; and S3, analysis and judgment of a detection result: analyzing and judging whether the sample to be detected is infected with babesia gibsoni and / or babesia canis according to a Ct value in a fluorescent quantitative PCR reaction system. 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 present invention relates to the technical field of pathogen detection of canine babesiosis, and in particular to a detection method for distinguishing Babesia canis from Babesia gibsoni for non-disease diagnosis purposes. Background Art

[0002] Canine babesiosis is caused by the parasite Babesia, a tick-borne blood-borne protozoan, that infects canine red blood cells. Infection in dogs can lead to fever and lethargy, followed by hemolytic anemia, red blood cell rupture, coagulation abnormalities in the liver, lungs, and kidneys, electrolyte imbalances, and organ dysfunction. The disease typically presents an acute course and poses a serious threat to the dog's health.

[0003] At present, the occurrence and distribution of canine babesiosis in my country are 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 primary pathogens causing canine babesiosis in my country are Babesia canis and Babesia gibsoni. Babesia canis is relatively large, typically 4-5 μm in length. Babesia canis vogeli, Babesia canis, and Babesia canisrossi are the three most common species of canine Babesia in my country. Babesia gibsoni is very small, measuring 1-2.5 μm in length. It is the primary causative species of babesiosis in my country. Clinically, the clinical symptoms caused by canine Babesia and Babesia gibsoni are similar, but there are differences in drug treatment. Triazoguanidine and oxydiphenyl ether isethionate are highly effective against canine Babesia but cannot eliminate Babesia gibsoni. The combination of atovaquone and azithromycin is the most effective treatment for Babesia gibsoni. Therefore, effective differentiation of the 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 smearing and reading techniques, which places high demands on the operator. In addition, it is difficult to detect the worms in the early stages of infection, which can easily lead to missed detection.

[0006] With the development of molecular biology techniques, fluorescent quantitative PCR has been widely used in the diagnosis of canine babesiosis due to its ultra-high sensitivity and specificity. However, there is currently no method that can effectively distinguish between Babesia gibsoni and Babesia canis. Furthermore, the preparation of existing fluorescent quantitative PCR reagents is cumbersome, requiring specialized PCR laboratories, and the long detection cycle of approximately 2 hours, limiting its widespread use in pet hospitals.

[0007] Therefore, the existing technology needs to be further improved. Summary of the Invention

[0008] To address the above problems, the present invention provides a primer-probe combination, a detection kit, and a detection method that can quickly and accurately distinguish between Babesia canis and Babesia gibsoni. The detection method is simple to operate, can quickly and effectively distinguish between Babesia gibsoni and 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] 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 reconstitution 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 as follows: 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 35–45 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.

[0010] The above-mentioned detection method for distinguishing Babesia canis from Babesia gibsoni is simple to operate, highly specific, and accurate, and significantly shortens the detection cycle. The total time from sample addition to report issuance can be reduced to 20-30 minutes, offering the advantages of a short cycle and high efficiency.

[0011] Optionally, the S2 step is performed on an ultra-fast fluorescence quantitative PCR instrument.

[0012] 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.

[0013] Further preferably, in step S2, the ultra-fast fluorescent quantitative PCR amplification program is: 90° C., 2 s; 65° C., 5 s; and 40 cycles of amplification.

[0014] Preferably, in step S3, intelligent result analysis and report interpretation software is used. The software includes a database of various pre-stored common items and can automatically identify the fluorescence quantitative PCR results to generate an interpretation report. In practical applications, this is more convenient for rapid display of experimental results.

[0015] Optionally, in the detection method, the specific analysis and judgment method of step S3 is: If the fluorescent channel of the fluorescent probe Jiba-P is the first fluorescent channel, the fluorescent channel used by the fluorescent probe Dogba-P is the second fluorescent channel; When the Ct value of the first fluorescence channel is <40 and there is no Ct value in the second fluorescence channel, it indicates 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 indicates 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 fluorescence channel and no Ct value in the second fluorescence channel, it indicates that there is no Babesia gibsoni and Babesia canis in the sample.

[0016] Optionally, in the detection method, if the fluorescent channel used by the fluorescent probe ACTB-P is the third fluorescent channel, then when the Ct value of the third fluorescent channel is less than 40, it indicates that the sample sampling is qualified.

[0017] Further preferably, the first fluorescent channel is FAM, the second fluorescent channel is HEX, and the third fluorescent channel is CY5.

[0018] Optionally, the lyophilized PCR reagent is prepared by lyophilizing a PCR mixture, wherein the PCR mixture comprises: the primer-probe combination, DNA polymerase, dNTPs, a buffer, and a lyoprotectant. 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, 1 μl dNTPs, and 12.5 μl lyoprotectant.

[0019] The primer-probe composition comprises: Primers and probes for detecting the specific gene TRAP of Babesia gibsoni: upstream primer Giba-F with a sequence shown in SEQ ID NO.1, downstream primer Giba-R with a sequence shown in SEQ ID NO.2, and fluorescent probe Giba-P with a sequence shown in SEQ ID NO.3; Primers and probes for detecting 16S rRNA of Babesia canis: upstream primer Canis-F with a sequence shown in SEQ ID NO. 4, downstream primer Canis-R with a sequence shown in SEQ ID NO. 5, and fluorescent probe Canis-P with a sequence 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.

[0020] 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.

[0021] Optionally, 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.

[0022] Preferably, the lyoprotectant consists of the following ingredients: 4% W / V% trehalose and 3% W / V% dextran.

[0023] 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.

[0024] Preferably, the DNA polymerase is super Taq DNA polymerase. The super Taq DNA polymerase is a modified and optimized Taq DNA polymerase with better tolerance to inhibitors and higher amplification and extension efficiency.

[0025] Optionally, 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.

[0026] Preferably, the PCR denaturation accelerator is 1-15% V / V% DMSO. More preferably, the PCR denaturation accelerator is 15% V / V% DMSO.

[0027] Preferably, the PCR mixture further comprises: a nucleic acid release agent; the nucleic acid release agent comprises the following components at the following concentrations: 100 mM KOH, 5 mM EDTA, 100 mM Tris-HCl and 5% V / V% sorbitol; Alternatively, the reconstitution solution is a sterile aqueous solution containing 5% v / v% glycerol.

[0028] Optionally, the freeze-dried PCR reagent is prepared by mixing all the components and then freeze-drying them through a multi-stage freeze-drying process, wherein the multi-stage freeze-drying process is: Pre-freezing stage: -45°C, hold for 2 hours; primary sublimation drying: raise the temperature to -30°C while simultaneously turning on the vacuum pump to rapidly evacuate to 1 mbar, hold for 2 hours; then raise the temperature to -15°C, hold for 2 hours; secondary sublimation drying: continue heating to 10°C, hold for 2 hours; then raise the temperature to 25°C, hold for 2 hours. Capping: After the freeze-drying process is completed, nitrogen is flushed to equilibrate to atmospheric pressure, and then eight rows of tubes are capped and sealed to complete the freeze-drying process.

[0029] The detection kit for distinguishing between Babesia canis and Babesia gibsoni also includes: a positive quality control and a negative quality control, wherein the positive quality control is a mixture of Babesia gibsoni plasmid, Babesia canis plasmid and canine ACTB internal reference plasmid, with the ratio of the three being 1:1:1; the negative control is sterile deionized water.

[0030] The present invention has the following beneficial effects: 1. The present invention achieves, for the first time, the simultaneous and accurate detection of Babesia gibsoni and Babesia canis in the same PCR reaction system, and effectively distinguishes Babesia gibsoni from Babesia canis based on their specific gene sequences. This provides a precise medication regimen for dogs infected with Babesia, is of great significance for the prevention and treatment of canine babesiosis, and has a significant promoting effect on the healthy development of my country's dog breeding industry.

[0031] 2. The present invention uses direct release of blood samples, fully premixed freeze-dried PCR reagents, ultra-fast nucleic acid amplification procedures and a supporting ultra-fast fluorescent quantitative PCR detection and analysis system. The equipment is compact 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 sample addition to report issuance, it takes ≤30 minutes, which is conducive to its promotion and use in pet hospitals.

[0032] 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 contamination. 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

[0033] Figure 1The amplification curves of the three sets of primers and probes for the present application, comparative example 1, and comparative example 2, respectively, are obtained when the positive plasmids are amplified. 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 amplify the samples of Babesia gibsoni; 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 sample; wherein Figure 3 A is the result of amplifying Babesia canis samples using the canine primers among the three sets of primer probes; Figure 3 B is the result of amplifying Babesia canis samples using the Giba primers among the three sets of primer probes; Figure 4 The kit for detecting Babesia canis 1×10 1 ~1x10 6 The results of fluorescence quantitative amplification of the serial dilution plasmid with 10 copies / ul; Figure 5 The kit of Example 4 detects Babesia gibsoni 1×10 1 ~1x10 6 The results of fluorescence quantitative amplification of the plasmid with gradient dilution of copies / ul; Figure 6 The kit for detecting the canine reference gene ACTB 1x10 1 ~1x10 6 The fluorescence quantitative amplification results of the plasmid with gradient dilution of copies / ul. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a 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 of 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 this field. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0035] Example 1 Screening of primer-probe combinations and kit setup 1. Design of primer-probe combination Forty TRAP gene sequences of Babesia gibsoni were obtained from NCBI and aligned using Clustal X2 software to identify relatively conserved gene sequences. Multiple pairs of upstream and downstream primers and probes were designed to specifically amplify Babesia gibsoni.

[0036] Forty Babesia canis 16s rRNA sequences and twenty Babesia gibsoni 16s rRNA sequences were obtained from NCBI. Sequence alignment was performed using Clustal X2 software to identify multiple gene sequences that were relatively conserved in Babesia canis but specific to Babesia gibsoni. Multiple pairs of upstream and downstream primers and probes were designed accordingly.

[0037] The canine ACTB gene sequence was obtained from NCBI and used as an internal reference gene to design upstream and downstream primers and probes. Primers for this internal reference gene were used to avoid false negatives from interfering with the test results. In other embodiments, other conserved canine genes may also be selected as internal reference genes.

[0038] 2. Screening and Validation of Primer-probe Combinations Through the combination verification and screening of primer probes using multiple samples and plasmids, a primer probe combination with good specificity, high sensitivity, and high amplification efficiency for simultaneously distinguishing Babesia canis and Babesia gibsoni was screened out. The specific primer probe information is shown in Table 1 below: Table 1 Primer and probe sequences for Babesia gibsoni, Babesia canis, and ACTB genes

[0039] 3. Preparation of the Kit This embodiment designs and applies a kit for the multiple sets of primers and probes in Table 1 above.

[0040] As shown in Table 2, the kit includes: freeze-dried PCR reagents, nucleic acid release agent, reconstitution solution, positive quality control and negative control.

[0041] Among them, the freeze-dried PCR reagent includes the following components: 0.5μM Giba-F, 0.5μM Giba-R, 0.25μM Giba-P, 0.5μM Dogba-F, 0.5μM Dogba-R, 0.25μM Dogba-P, 0.4μM ACTB–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 protectant, and is divided into eight PCR tubes and then freeze-dried according to a multi-stage freeze-drying program.

[0042] The freeze-drying process is as follows: pre-freeze at -45°C for 2 hours; primary sublimation drying: heating to -30°C while simultaneously pumping the vacuum pump to 1 mbar for 2 hours; then heating to -15°C for 2 hours; secondary sublimation drying: heating to 10°C for 2 hours; and finally heating to 25°C for 2 hours. Capping: After the freeze-drying process is complete, nitrogen is introduced to balance the pressure to atmospheric pressure, and then eight tubes are capped and sealed in a row to complete the freeze-drying process.

[0043] 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.

[0044] The nucleic acid releaser is used for releasing nucleic acid from a blood sample, and contains the following components at the following concentrations: 100 mM KOH, 5 mM EDTA, 100 mM Tris-HCl, and 5% v / v% sorbitol.

[0045] The reconstitution solution is used to reconstitute the entire premixed lyophilized reagent, which contains 5% v / v% glycerol in sterile deionized water.

[0046] The positive quality control is a mixture of a Babesia gibsoni 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 gibsoni plasmid is shown in SEQ ID NO. 10 in the sequence listing, the target gene of the Babesia canis plasmid is shown in SEQ ID NO. 11 in the sequence listing, and the target gene sequence of the ACTB plasmid is shown in SEQ ID NO. 12 in the sequence listing.

[0047] The lyoprotectant consists of the following components: 4% W / V% trehalose and 3% W / V% dextran.

[0048] Table 2 Composition of the kit

[0049] 4. This embodiment also provides a detection method for distinguishing Babesia canis from Babesia gibsoni using the above method, which comprises the following steps: S1. Draw 1 ml of canine venous blood using an EDTA blood collection tube; and remove all reagents from 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 mixture and add it to the reconstitution solution, vortex and shake to mix, take out 25ul and add it to the lyophilized reagent.

[0050] At the same time, add 25ul of positive control and negative control to the corresponding lyophilized reagents as positive and negative controls. Cap the tubes, mix well, and centrifuge.

[0051] S2. Place the centrifuged reaction mixture into a rapid fluorescence quantitative PCR detection and analysis system (i.e., an ultra-rapid fluorescence quantitative PCR instrument) and perform ultra-rapid fluorescence quantitative PCR amplification according to the following amplification procedure; The ultra-fast fluorescence quantitative PCR amplification program was as follows: 90°C, 2s; 65°C, 5s (fluorescence collection); and 40 cycles of amplification were performed.

[0052] 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.

[0053] The specific analysis and judgment methods are as follows: (as shown in Table 3).

[0054] When the Ct value of the FAM channel is <40 and there is no Ct value in the HEX channel, it indicates 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 indicates 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 gibsoni and Babesia canis in the sample; When the Ct value of the CY5 channel is less than 40, it indicates that the sample is qualified.

[0055] In other embodiments, the above three fluorescent channels may be replaced with other types of fluorescent channels, and the solution of the present invention is not limited to the specific configuration of this embodiment.

[0056] Table 3 Analysis and judgment methods

[0057] Note: If there is no Ct value for the CY5 channel, retesting is required.

[0058] Example 2 Comparison of the effects of primer-probe combinations 1. Experimental methods This experiment provides kits for Comparative Examples 1, 2, 3, and 4. These kits differ from the kit of Example 1 in that they utilize different primer-probe combinations, as detailed in Table 1. The fluorescent probes on the primer probes in each control group were configured similarly to those in Example 1 for comparative analysis. The kit preparation methods for each group were the same as those in Example 1.

[0059] Table 4 Primer probes for the control group

[0060] The kits prepared in Example 1 of the present application, Comparative Examples 1, 2, 3, and 4 were respectively used according to the detection method of Example 1, and the common amplification procedure of 95°C for 5 min; (95°C for 15 s, 60°C for 30 s (collecting fluorescence), 40 cycles) was used to perform the following experiment on the same sample: (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.

[0061] (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.

[0062] Compare the experimental results of each kit.

[0063] 2. Experimental results and analysis like Figure 1 As shown, from the results of amplifying the positive plasmids 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 Giba 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 increases). The amplification of the internal reference ACTB in Comparative Examples 1 and 2 is slightly inhibited.

[0064] Figure 2 The results of five sets of primers amplifying Babesia gibsoni samples are shown in Figure 2. Figure 2 As shown in Figure A, the results of amplifying Babesia gibsoni samples using five sets of primers using the Canis nuclei primers showed that the Canis nuclei primers of Comparative Examples 1, 3, and 4 had non-specific amplification on Babesia gibsoni samples, while the Canis nuclei primers of Comparative Example 2 and Example 1 had no non-specific amplification on Babesia gibsoni. Figure 2 As shown in Figure B, the results of amplifying Babesia gibsoni samples using the five primer sets of Giba showed that all five primer sets could amplify Babesia gibsoni, and the Ct values ​​of Comparative Examples 1, 3 and 4 were earlier than those of Comparative Example 2 and Example 1.

[0065] Figure 3 The results of five sets of primers amplifying Babesia canis samples are shown in Figure 2. Figure 3 As shown in A, all five primer sets can amplify the dog sample, among which the amplification Ct values ​​of Comparative Examples 1, 3 and 4 are earlier, and Comparative Example 2 is the same as Example 1. Figure 3As shown in Figure B, the five sets of primers from Gibbs were used to amplify the Babesia canis sample. Comparative Example 1 had non-specific amplification of the Babesia canis sample, while Comparative Examples 2, 3, 4 and Example 1 had no non-specific amplification.

[0066] 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 between Babesia gibsoni and 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 between Babesia gibsoni and Babesia canis. Therefore, Examples 1 and 2 are preferred.

[0067] Example 3 Optimization of the amplification procedure in the detection method of this application 1. Test method 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 lupus and Gibberella positive was used for comparative verification.

[0068] Procedure 1: Common fluorescence PCR amplification procedure, pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s (fluorescence collection), 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 seconds, annealing at 60°C for 5 seconds (fluorescence collection), 40 cycles; Program 4: denaturation at 90°C for 2 seconds, annealing at 65°C for 5 seconds (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 on the basis of Program 1, which has little effect on the kit. Program 3 lowers the denaturation temperature to 90°C. 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.

[0069] 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.

[0070] Table 5 Comparison of amplification by different programs

[0071] 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, and 15% v / v% DMSO.

[0072] B: Prepare reagent B by adding 0.5 μM Giba-F, 0.5 μM Giba-R, 0.25 μM Giba-P, 0.5 μM Dogba-F, 0.5 μM Dogba-R, 0.25 μM Dogba-P, 0.5 μM ACTB–F, 0.5 μM ACTB–R, 0.25 μM ACTB–P; 0.6 μL Taq DNA polymerase, 2.5 μL 10x buffer, 1 μL dNTPs, and 10% V / V% DMSO.

[0073] C: Prepare reagent C by adding 0.5 μM Giba-F, 0.5 μM Giba-R, 0.25 μM Giba-P, 0.5 μM Dogba-F, 0.5 μM Dogba-R, 0.25 μM Dogba-P, 0.5 μM ACTB–F, 0.5 μM ACTB–R, 0.25 μM ACTB–P; 0.6 μL Taq DNA polymerase, 2.5 μL 10x buffer, 1 μL dNTPs, and 5% V / V% DMSO.

[0074] D: Prepare reagent D by adding 0.5 μM Giba-F, 0.5 μM Giba-R, 0.25 μM Giba-P, 0.5 μM Dogba-F, 0.5 μM Dogba-R, 0.25 μM Dogba-P, 0.5 μM ACTB–F, 0.5 μM ACTB–R, 0.25 μM ACTB–P; 0.6 μL Taq DNA polymerase, 2.5 μL 10x buffer, 1 μL dNTPs, and 15% v / v% formamide.

[0075] E: Prepare reagent E by adding 0.5 μM Giba-F, 0.5 μM Giba-R, 0.25 μM Giba-P, 0.5 μM Dogba-F, 0.5 μM Dogba-R, 0.25 μM Dogba-P, 0.5 μM ACTB–F, 0.5 μM ACTB–R, 0.25 μM ACTB–P; 0.6 μL Taq DNA polymerase, 2.5 μL 10x buffer, 1 μL dNTPs, and 15% W / V% betaine.

[0076] The ultra-fast fluorescence quantitative PCR amplification program was as follows: 90°C, 2s; 65°C, 5s (fluorescence collection); and 40 cycles of amplification were performed.

[0077] (2) Loading samples onto the tester: Follow the operating procedures of Example 1 and use two known positive blood samples for comparative verification.

[0078] 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 was significantly delayed, affecting the detection limit (i.e., sensitivity) of the product. The sample amplification of Reagent C was not observed, and the Ct values ​​of the sample amplification of Reagent D and Reagent E were slightly delayed. Therefore, Reagent A is preferred.

[0079] Table 6 Optimization of different denaturants

[0080] 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, and anti-interference were performed.

[0081] (1) Sensitivity test ① Experimental method: The synthetic plasmid standards of Babesia canis, Babesia gibsoni and the synthetic plasmid standards of the canine internal reference gene ACTB were serially diluted to a concentration of 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.

[0082] ② Experimental results and analysis A. The experimental results show that the kit has 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 1x101 ~1x10 6 The minimum detection limit of the plasmid was 1x10 copies / ul. 1 Each minimum detection limit standard was tested 20 times, and the positive detection rate was 100%. The verification results are shown in Table 7 below.

[0083] B. Figure 5 The above kit was used to detect Babesia gibsoni 1x10 1 ~1x10 6 The results of fluorescence quantitative amplification of the gradient dilution plasmid of copies / ul show that the detection sensitivity of the plasmid to Babesia gibbsii is high, and 1x10 1 copies / ul.

[0084] C. Figure 6 The above kit was used to detect the canine reference gene ACTB 1x10 1 ~1x10 6 The results of fluorescence quantitative amplification 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.

[0085] Table 7 Sensitivity test results

[0086] (2) Precision (repeatability) ① Experimental method: 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.

[0087] ②Experimental results: As shown in Table 8, the standard deviations of the Ct values ​​for different concentrations of Babesia gibsoni were 0.21 and 0.30, respectively, with coefficients of variation of 0.80% and 0.89%, respectively. The standard deviations of the Ct values ​​for different concentrations of Babesia canis were 0.03 and 0.25, respectively, with coefficients of variation of 0.13% and 0.75%, respectively. The standard deviations of the Ct values ​​for different concentrations of the canine internal reference, ACTB, were 0.16 and 0.38, with coefficients of variation of 0.58% and 1.12%, respectively. These results demonstrate that the kit has good reproducibility for the detection of Babesia gibsoni, Babesia canis, and the canine internal reference, ACTB.

[0088] Table 8 Precision verification results for Babesia gibsoni, Babesia canis, and the canine internal reference ACTB

[0089] (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 gibbsii samples C1, C2, C3, C4 and C5, which were confirmed positive by sequencing, to evaluate the accuracy of the kit.

[0090] 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.

[0091] Table 9 Accuracy test results of the kit

[0092] (4) Specificity ① Experimental methods The above kit was used to test negative reference samples N1-N8 (samples confirmed by sequencing to be negative for Babesia canis and Babesia gibbsii, and positive for other pathogens) to evaluate the analytical specificity of the kit. The detection method was similar to that in Example 1. The negative reference samples N1 were Leptospira, N2 were Toxoplasma, N3 were Haemophilus, N4 were Hepatozoon, N5 were Anaplasma phagocytophilum, N6 were Ehrlichia canis, N7 were Borrelia burgdorferi, and N8 were Heartworm.

[0093] ②Experimental results: The experimental results showed that the negative reference samples N1 to N8 were all negative, with no cross-contamination. This result demonstrates the high specificity of the test kit.

[0094] (5) Anti-interference ① Experimental methods The kit of the present invention was used to detect common interfering substances in blood that can inhibit PCR, including 60 g / L albumin, 37 mmol / L triglycerides, 342 μmol / L bilirubin, and 200 g / L hemoglobin. The interfering substances were added to positive samples for testing. The anti-interference ability of the kit was evaluated.

[0095] ②Experimental results: The experimental results are shown in Table 10. The common interfering substances in blood (albumin, triglycerides, bilirubin and hemoglobin) mentioned above did not interfere with the test results. This result shows that the detection kit has strong anti-interference ability.

[0096] Table 10

[0097] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solutions and concepts of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for distinguishing Babesia canis from Babesia gibsoni for non-disease diagnosis purposes, characterized in that: The detection method comprises the following steps: S1. Sample processing: Add the sample to the nucleic acid release agent, vortex and shake, pipette the suspension and add it to the reconstitution solution, shake and mix, add the mixture to the freeze-dried PCR reagent, mix and centrifuge to obtain the 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 as follows: 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 35–45 cycles; S3. Analysis and judgment of test results: judging whether the sample to be tested is infected with Babesia gibsoni and / or Babesia canis based on the Ct value analysis in the fluorescent quantitative PCR reaction system; The freeze-dried PCR reagent is prepared by freeze-drying a PCR mixture, wherein the PCR mixture comprises a primer-probe combination, a DNA polymerase, dNTPs, a buffer and a freeze-drying protective agent; The primer-probe composition includes: an upstream primer Jiba-F whose sequence is shown in SEQ ID NO.1, a downstream primer Jiba-R whose sequence is shown in SEQ ID NO.2, and a fluorescent probe Jiba-P whose sequence is shown in SEQ ID NO.3; an upstream primer Canine-F whose sequence is shown in SEQ ID NO.4, a downstream primer Canine-R whose sequence is shown in SEQ ID NO.5, and a fluorescent probe Canine-P whose sequence is 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 detection method according to claim 1, wherein The specific analysis and judgment method of step S3 is: If the fluorescent channel of the fluorescent probe Jiba-P is the first fluorescent channel, the fluorescent channel used by the fluorescent probe Dogba-P is the second fluorescent channel; When the Ct value of the first fluorescence channel is <40 and there is no Ct value in the second fluorescence channel, it indicates 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 indicates 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 fluorescence channel and no Ct value in the second fluorescence channel, it indicates that there is no Babesia gibsoni and Babesia canis in the sample.

3. The detection method according to claim 2, characterized in that The primer-probe composition further includes 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.

4. The detection method according to claim 1, wherein The fluorescent reporter group is selected from FAM, HEX, CY5, ROX, and VIC; the fluorescent quencher group is selected from BHQ1, BHQ2, and BHQ3.

5. The detection method according to claim 2, characterized in that The nucleic acid releasing agent includes the following components at the following concentrations: 100 mM KOH, 5 mM EDTA, 100 mM Tris-HCl, and 5% W / V% sorbitol.

6. The detection method according to claim 2, characterized in that The PCR mixture is mainly prepared from the following components: 0.1-1 μM primer probe, 0.2-0.8 μl super Taq DNA polymerase, 2.5 μl 10x buffer, 1 ul dNTPs, and 12.5 μl lyophilized protective agent.

7. The detection method according to claim 1, characterized in that The preparation method of the freeze-dried PCR reagent comprises the following steps: mixing all the components and then freeze-drying them through a multi-stage freeze-drying process, wherein the multi-stage freeze-drying process is: Pre-freezing stage: -45°C, hold for 2 hours; primary sublimation drying: raise the temperature to -30°C while simultaneously turning on the vacuum pump to rapidly evacuate to 1 mbar, hold for 2 hours; then raise the temperature to -15°C, hold for 2 hours; secondary sublimation drying: continue heating to 10°C, hold for 2 hours; then raise the temperature to 25°C, hold for 2 hours. Capping: After the freeze-drying process is completed, nitrogen is flushed to equilibrate to atmospheric pressure, and then eight rows of tubes are capped and sealed to complete the freeze-drying process.

8. The detection method according to claim 6, characterized in that The lyoprotectant consists of the following components: 4% W / V% trehalose and 3% W / V% dextran.

9. The detection method according to claim 1, wherein The PCR mixture also includes: a PCR denaturation accelerator, which 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; the reconstitution solution is a sterile aqueous solution containing 5% V / V% glycerol.

Images