TWI532844B - Method of distinguishing dog coccidia - Google Patents

Description

Method for distinguishing the classification of canine echinococcosis

The present invention relates to a method of distinguishing animal pathogenic microorganisms, and more particularly to a method for distinguishing the classification of canine echinococcosis.

With the trend of declining birth and aging, more and more pets have entered the family and become the most important "companion animals" of modern people. According to statistics, it is estimated that the pet-related market in Taiwan is as high as 25 billion yuan a year. The increase in pets is more and more demanding than veterinarians. Animal medicine is becoming increasingly important.

At present, the proportion of pet dogs accounts for more than half of the total pets. As the population of pets increases, the prevalence of veterinary hospitals also increases. Since pets are susceptible to damage to body tissues and even deaths after being infected by certain pathogens, it is extremely important to grasp the golden period of disease diagnosis. The veterinary market is also in urgent need for rapid detection. For the detection of pathogenic bacteria, a good fast screening or rapid inspection mode has been established in the human medical system, but the establishment of the veterinary system is not perfect. At this stage, only the pathogens of a few cats and dogs are developed, such as the dog's four-in-one. Fast screening kit (heartworm, Lyme disease, canine/horse type Ehrlich, platelet type Ehrlich), cat's triple play (cat immunodeficiency virus, feline leukemia virus, silk) Therefore, the rapid screening kit or rapid test mode for establishing other infectious diseases of dogs and cats has become the focus of today's veterinary system.

Coccidiosis is a blood-related disease caused by blood parasites, called parasites, a "protozoa" that causes progressive anemia in dogs. The media is mainly Tick bites, and a small part is due to blood transfusions and infections through the placenta to the fetus.

Symptoms after infection may take about 10-20 days. There are often no obvious symptoms in the initial stage. There is only a lack of appetite. However, the main function of red blood cells is to carry oxygen. If infected by parasites, the oxygen-carrying function will decrease. This leads to a gradual deterioration of mental status, which causes phagocytic cells of autologous white blood cells to devour their own red blood cells. Dogs have different responses and symptoms after infection, and are mainly classified into non-specific, chronic and acute infections. Non-specific infection, more symptoms, will There are ascites, gastrointestinal symptoms, neurological symptoms, peripheral edema, and heart and lung disease. In chronic infection, dogs have intermittent fever, loss of appetite and weight loss; acute infections may have elevated body temperature, pale mucous membranes, blood spots, jaundice, hepatosplenomegaly, and even acute death.

At present, there are more than one hundred kinds of cokeworms. The infected dogs are mainly divided into two types: large and small worms. The large worms are also known as Babesia canis . The small worms are also known as Gibbs pine worms. Babesia gibsoni ). When a coccidiosis infection occurs, usually only one of them occurs. It is not a large cokeworm or a small cokeworm. There are few mixed infections. Because of differences in medication, careful and rapid identification is required to avoid delaying the disease. , causing subsequent hard to cure, and there may be recurrence. There is no fast screening or rapid detection method for the detection of coccidia, so it is imperative to develop rapid and accurate detection technology.

The traditional method of detecting insects is to use blood smears. Whether it is large worms or small worms, specific translucent rings appear around the black spots in the red blood cells. Usually, a black dot is found in the red blood cells to represent Babesia gibsoni. Infection, and the double pear-shaped black spots are characteristic of Babesia canis infection. However, the probability of finding pathogens in this way is not high, generally it is a period of onset, and it is only visible when the number of coccidia accumulates a certain amount.

Early coccidiosis infections are more difficult to find from blood smears, and traditional PCR techniques detect that their primers may bind to non-coke DNA (ie, the patient's own DNA), causing false positives. The production of Nested PCR (nested PCR) technology has been applied to the detection of coccidia by those skilled in the art. Nested PCR mainly uses two pairs of primers for two rounds of PCR amplification reactions. The first round of amplification is no different from traditional PCR. The product after the first round of PCR is specifically amplified by the second pair of primers, and the probability of replication to DNA other than the coccidia is reduced by two PCR amplifications. The detection sensitivity has increased by 10 ² ~ 10 ³ times, but because of the cross-infection of 2 PCRs, it is still impossible to accurately classify and avoid false positives.

The disadvantage of Nested PCR is that the experimental procedure is cumbersome. The result cannot be directly seen after PCR. It is necessary to further confirm the presence or absence of the cDNA of the cokeworm by electrophoresis, and because the 18S rDNA gene sequences of the large and small worms are too close, only a few The bases are different, so although two different sets of primers are designed to distinguish, it is not necessary to see the type on the electropherogram, and a nucleic acid sequencer or It is a restriction enzyme RFLP confirmation, so it takes at least one day to get an inspection report. In the case of time-consuming and accurate, it is necessary to develop faster and more accurate detection methods to improve the quality of the coroner test.

As mentioned above, considering the needs of the market and the problems of today's detection technology, it is urgent to establish a fast and accurate dog pyrotype detection technology to meet the needs of veterinary diagnosis timeliness. Other models of rapid detection of pathogens on the veterinary system will follow suit.

Accordingly, the present invention provides a method for distinguishing a dog phobia typing, the steps comprising: (a) providing a nucleic acid sample; (b) performing a certain amount of immediate polymerase chain reaction on the nucleic acid sample (Q real-time) PCR); and (c) performing a melting curve analysis to distinguish the canine worm typing of the nucleic acid sample; wherein the canine genus classification includes Babesia canis (BC) and Kyrgyzstan Babesia gibsoni (Bg), and the primer used in the real-time quantitative polymerase chain reaction pair SEQ ID NO: 3 and SEQ ID NO: 4 (BCBghsp231F/R) for encoding heat shock proteins in the nucleic acid sample A segment of the 70 gene.

Preferably, wherein step (c) further comprises performing a high-resolution melt-melting (HRM) analysis.

Preferably, the nucleic acid sample is extracted from a blood sample of a body; preferably, the blood sample is a blood cell sample after separation by blood cell plasma.

Preferably, wherein step (C) further comprises using LC green dye, SYBR green dye or SYTO 9 dye.

Through the technical means adopted by the present invention, two kinds of worms can be successfully performed by high resolution melting method using the heat shock protein 70 gene of canine worm and Gibbs pine worm. Distinguish and use the Ct value of the positive control group with known concentration to determine the concentration of the test insect (up to absolute quantitation), providing a rapid detection method (including nucleic acid extraction and The HRM time (a total of about four hours) greatly provides the prime time for the treatment of clinical veterinarians. In the future, the technical contents and advantages thereof disclosed in the present invention can be utilized, including improving the detection accuracy and reducing the advantages of false positive occurrence, thereby establishing a rapid detection system for large and small size insects, and providing a more efficient detection service.

First, the specific implementation steps of the present invention are briefly described. The present invention provides a method for distinguishing the classification of canine echinococcosis, the steps comprising: (a) providing a nucleic acid sample; and (b) performing a certain amount of immediate polymerase on the nucleic acid sample. a chain reaction (Q real-time PCR); and (c) performing a melting curve analysis to distinguish a canine worm typing of the nucleic acid sample; wherein the canine genus typing includes a canine worm ( Babesia canis , BC) and Babesia gibsoni (Bg), and the primer used in the real-time quantitative polymerase chain reaction is directed against SEQ ID NO: 3 and SEQ ID NO: 4 (BCBghsp231F/R) A segment of the heat shock protein 70 gene is encoded in the nucleic acid sample. Preferably, the step (c) further comprises performing a high-resolution melt-melting (HRM) analysis; the nucleic acid sample is extracted from a blood sample of the body; the blood sample is a blood cell separated by blood cell plasma. Sample; wherein step (C) further comprises using LC green dye or SYBR green dye. The above is only a brief implementation step, and the specific implementation details and experimental results are detailed below.

Experimental material method Sample collection and nucleic acid extraction

A total of 80 clinical blood samples from animal hospitals in Taiwan were collected and stored in the EDTA anticoagulation tube at a low temperature (4 °C) for delivery to the company for pre-treatment (plasma hematocrit) for the purpose of experimentation. Sexually large, may see anemia, hemolysis, dehydration, etc., so plasma blood cell separation before nucleic acid extraction, reduce the difference in nucleic acid extraction concentration, and increase the concentration of worm nucleic acid contained in the sample to avoid false positive occur.

Plasma blood cell separation method (pretreatment) and automatic nucleic acid extraction: plasma blood cells are centrifuged at 3000 rpm for 15 minutes, and then 200 μL of the lower blood cells are taken in the first and seventh rows of the 96-well disk, and Add nucleic acid extraction related reagents to each reaction row (500 μL lysis buffer in the first and seventh rows, 50 μL magnetic beads and 750 μL washing buffer A in the second and eighth rows, and 750 μL washing buffer A in the third and ninth rows, Add 750μL washing buffer B to the fourth, fifth and tenth and eleventh rows, add 50μL eluting buffer to the sixth and twelve rows, insert the magnetic rod sleeve, and press the start button to start the blood cell nucleic acid extraction.

In the thermal melting curve (melting curve) analysis method of distinguishing canine Babesia (Babesia canis, BC) and Jibu Song Babesia (Babesia gibsoni, Bg)

Using the highly differential sequence characteristics of BC and Bg heat shock protein (hsp) 70, the primer pair BCBghsp231F/R was designed (Fig. 13). The DNA fragment encoding heat shock protein 70 is SEQ ID NO: 1 and SEQ ID NO: 2, the GC content of the sequence on BC and Bg was 55.41% and 48.48%, respectively, and the thermal dissolution curve temperature value of BC was expected to be greater than Bg. The amount of PCR reaction reagent was 10 μL of 2X Taq premix, 1.2 μL of 25 mM MgCl ₂ , 1 μL of DMSO, 2 μM of BCBghsp231F/R 2 μL, 10 μL of 10 μM template, 0.8 μL of DDW, denaturation at 95 ° C for 5 minutes, 95 At 35 ° C, 58 ° C, 72 ° C for 30 seconds, a total of 35 cycles, 72 ° C, 5 minutes to extend the reaction to the target gene product amplification reaction, take 5 μL of the product after PCR reaction, add SYBR green 5μL in a 384-well reaction plate, set the experiment The mode is "standard curve" and includes the reaction conditions of the melt curve stage after the PCR reaction: 95 ° C, 15 seconds, 60 ° C, 1 minute, 95 ° C, 15 seconds, detecting the firefly at a temperature increase rate of 0.05 ° C per second. Light value.

The distinction between Babesia canis (BC) and Babesia gibsoni (Bg) was performed using a High-Resolution Melting (HRM) analyzer (HR-1, Idaho Technology).

Since the fluorescent light required for HR-1 is LC green, it can be added before or after the PCR reaction. In the cost consideration, after the reaction, it is confirmed by electrophoresis that the product is increased, and then LC green is added. In addition, the PCR reagent used in the 2x Taq premix is containing a loading dye. In order to avoid the influence of the loading dye on the binding of LC green to the double-stranded DNA, the other does not contain the loading dye. The Taq is screened and confirmed to be a viable Taq before HRM.

1. Taq test: A total of three Taq tests were performed: (1) Power Taq (2) Hot Start (3) + (pfu plus), and each reaction volume was 20 μL per tube. PCR premix: PCR premix containing 2.5 mM MgCl ₂ , 0.2 mM dNTP, 0.2 u/μL Taq. A total of 6 samples (DNA template) were tested, 1. pBC (BC positive control), 2. pBg (Bg positive control), 3. BC with sample number 79, 4. Bg clinical sample (120419002) 5. Negative clinical specimen, 6.DDW, 2 μL primer pair (2uM BCBghsp231F/R) and 1 μL DNA template. PCR conditions: a denaturation reaction at 95 ° C, 5 minutes, 95 ° C, 58 ° C, 72 ° C for 30 seconds for a total of 35 cycles, 72 ° C, 5 minutes extended reaction to the target gene product amplification reaction. Electrophoresis conditions: 2% agarose containing 0.4 mg/mL EtBr at 250 V, 15 minutes later photographed under UV light and archived.

2. HR-1 analysis by LC green: 5 BC and Bg samples (DNA template), respectively, BC: 1.pBC (BC positive control), 2. Sample number 79, 3. Sample No. 40, 4. Unedited sample AM111008006 and 5. Sample number 57; Bg: 6.pBg (Bg positive control), 7. Unedited sample AM120419002, 8. Sample number 10, 9. Sample number 23 10. Sample No. 29, 11. Negative specimen, 12.DDW. Primer pair: 2uM BCBghsp231F/R. The PCR premix was added to 1 μL of the DNA template and 2 μL of the primer pair in the final volume of 20 μL as described above. The PCR conditions and running conditions are the same as above. HRM: 9 μL of the sample confirmed by electrophoresis and 1 μL of LC green was thoroughly mixed with a pipette, placed in a glass capillary (Light Cycler, USA) and centrifuged briefly, and the product to be tested was placed in the analyzer. The temperature is 0.3 ° C / sec, the initial fluorescence temperature is 60 ° C, and the termination temperature is 98 ° C.

3. After the determination is feasible, perform HR-1 analysis of BC and Bg samples (16 each, Table 4) by the above method.

4. Test SYBR green in HR-1 analysis: the tested sample is the same as 2., but it is 2x premix, so take 10μL of 2x SYBR green premix, add 1μL DNA template, 2μL primer pair and DDW 7μL, PCR The running conditions and the HRM method are the same as above.

Using ViiA ^TM The 7-high High-Resolution Melting (HRM) experimental model distinguishes between Babesia canis (BC) and Babesia gibsoni (Bg)

The BCBghsp231F/R primer pair with multiple base differences was tested. The test specimens of BC were 10 μg/μL pBC+ (BC positive control), sample numbers 47, 59, 63, 64, 66, 67; Bg test samples were 10 μg/μL pBg+ (Bg positive control), sample number 56, 54, 53, 51, 50, 49, wherein pBC and pBg were mixed with 1:1 and added to the test. The reagents used for the 2x Melt Doctor ^TM HRM reagent, formulated in a reaction volume of 10μL per tube of PCR premix: 5μL of 2x Melt Doctor ^TM HRM reagent (containing dye SYTO 9), 2uM 2μL of pair of primers, 1μL of test Sample, 2 μL of DDW. In 384-well plate positions are marked subject and obedient to the optical reaction film thereon after brief centrifugation (3000rpm, 1 min), placed ViiA ^TM 7 analyzer for analyzing a specimen, is set as the experimental model "High Resolution Melting" and includes a PCR reaction under the following conditions: Hold stage: 95 ° C, 10 minutes; PCR stage (40 cycles): 95 ° C, 15 seconds, 60 ° C, 1 minute; HRM stage: 95 ° C , 10 seconds, 60 ° C, 1 minute, 95 ° C, 15 seconds (0.025 ° C per second to detect a fluorescent value).

In addition, in order to quantify the concentration of the worm, a semi-sequence dilution (seim-dilute) was performed using a known 1:1 mixture of pBC and pBg (10 ug/uL) (Fig. 31-1, the obtained ct value was placed in the regression). The formula then obtains the concentration of the unknown sample.

Experimental result Sample collection and nucleic acid extraction

The clinical blood samples of animal hospitals in Taiwan were collected and stored in the EDTA anticoagulation tube at a low temperature (4 °C) and transported to the company for pre-treatment. Due to the diversity of clinical samples (anemia, hemolysis, dehydration, etc.), the difference in the nucleic acid extraction of the sample often increases the accuracy of the detection, so the characteristics of the parasites in the host red blood cells are utilized, and the blood cell separation method is adopted. Increasing the nucleic acid concentration of the worm to facilitate the experiment, as shown in Figure 2.

At present, 80 clinical samples have been collected, and the average concentration is 57.14 ng/μL, the ratio is 1.78. The concentration values of the 80 clinical samples are also different, which is presumed to be the cause of the sample itself. It may be infected specimens (white blood cells, neutrophils, mononuclear spheres, lymphocytes), regenerative anemia specimens (increased reticular balls), and low concentrations may be due to non-regenerative anemia, hemolysis specimens, etc.

The sample collection system described herein uses a method in which blood cells are separated from plasma, and blood is mainly composed of red blood. The ball and plasma make up 40% and 55% of the blood, respectively. Since the coccidia will be parasitic in the red blood cells, separating the blood cells from the plasma in the sample can reduce the interference of nucleic acid in the plasma, increase the concentration of the coccidial nucleic acid, and reduce the error in the test due to the too small concentration of the coccidio nucleic acid. In addition, the nucleic acid extraction utilizes the characteristics of good adsorption and stability of the magnetic beads, modifying the functional groups on the magnetic beads, and utilizing the adsorption of the functional groups and the nucleic acids to achieve the effect of rapidly obtaining nucleic acid and separation from the sample. The entire automated design not only saves time and costs, but also greatly improves the amount and purity of nucleic acids.

In the thermal melting curve (melting curve) mode quickly distinguish between canine Babesia (Babesia canis, BC) and Jibu Song Babesia (Babesia gibsoni, Bg)

In this experiment, the primers of the sequence nuclear test (BCBghsp231F/R) were designed by PCR using the high-difference characteristics of the heat shock protein 70 sequence, and the heat shock protein 70 product of the size-cavity was confirmed (Fig. 14). The product was then subjected to a thermal dissolution curve test result (Fig. 15), and the thermal dissolution curve temperature value was BC > Bg (Table 2).

The results of the distinction between Babesia canis (BC) and Babesia gibsoni (Bg) were performed using a High-Resolution Melting (HRM) analyzer (HR-1, Idaho Technology).

1. Taq test results are best with Power Taq (Figure 16).

2. The results of the PCR performed before the HR-1 analysis of the BC and Bg samples tested by LC green are shown in Fig. 17. The results of the HR-1 of the sample with the amplified product are shown in Fig. 18, and the BC can be initially known. The Tm value is greater than Bg.

3. After the test results of 1. and 2. above are feasible, the HR-1 test is further performed with LC green in BC and Bg clinical samples (Figures 19 and 20).

4. Test the results of SYBR green in HR-1 analysis (Figures 21, 22).

Using ViiA ^TM High-Resolution Melting (HRM) experimental model for the classification and quantification of Babesia canis (BC) and Babesia gibsoni (Bg)

The Ct and Tm values of the BC and Bg samples of the heat shock protein 70 gene tested in this experiment are shown in Table 5. The results of the heat shock protein 70 test: the amplification plot is shown in Figures 25 and 26, Raw melt curve. As shown in Figure 27, the derivative melt curve (derivative melt curve) The results are shown in Fig. 28. The results of the aligned melt curves are shown in Fig. 29 and the difference plot is shown in Fig. 30.

Quantitative results are shown in Figure 31-2 and Table 5. The ct values of the positive samples of each size were 6.24, 10.66, 14.52, 19.23, 22.97, 26.55, 29.62. The CCD value of the large coccidia positive test (BC+) was tested. 17.85, by using the known concentration of half-dilution (0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125 ug / uL) to obtain the regression curve formula (y = 5.686ln (x) + 2.7759), after the formula The concentration of the positive cokeworm positive sample tested was 0.00031191 ug/uL; the typing result is shown in Fig. 32. The Tm value of the positive test sample can confirm that the test sample is a large coke.

discuss

The following discussion can be obtained by the specific implementation methods disclosed above and the results obtained. The sample nucleic acid extraction method used in the above embodiments is a plasma blood cell separation method, which can increase the concentration of the insect body, avoid false negative results caused by anemia, hemolysis or low content of the worm.

Regarding the results of BC and Bg discrimination by heat shock protein 70, because of the long time taken by the conventional identification method, the heat shock protein 70 of BC and Bg is distinguished, and the heat shock protein 70 height of the two insect bodies is utilized. The characteristics of the difference are designed to be suitable for the primer pair. After the product is amplified, SYBR green is added to analyze the "melt curve" experimental mode in ViiA7 ^TM . The result can successfully distinguish BC and Bg by Tm value, but it is presented. The melting curve is rougher (there is a certain degree of difference with the actual HRM test results). Compared with the heat shock protein 70 directly in the "standard curve" experimental mode of ViiA7 ^TM (Applied Biosystems, USA), the method of analyzing the Tm value of BC and Bg samples is highly feasible, and the LC green and SYBR green tested are high. The results of the light were applied to the ViiA7 ^TM and the HR-1 (Idaho Technology) instrument which required the first increase of the product: LC green was better than the SYBR green in the HR-1 test (the obtained thermal melting curve was finer) Fluorescence can be added before or after the PCR reaction. It can be confirmed by electrophoresis to increase the product and then add fluorescence. This saves the cost of consumables. LC green is a 2x PCR premix that has been added to the enzyme, so it is a waste of enzyme. In terms of operation, HR-1 is only used once on the first specimen (glass capillary), and the operator must be on the side. After the analysis of the specimen, the next specimen is analyzed by human operation. The manpower is large, and on the ViiA7 ^TM operation, it is a 384 well reaction plate. Although it is better than the glass capillary, the advantage of high throughput is enough, and the instrument also has the amplification curve result to obtain the Ct value. Further conversion of the amount of insects (concentrated In the HRM analysis software, the Raw data, the derivative function diagram, the fluorescence signal normalization, and the difference plot including the melting curve are powerful functions, and the fluorescent light (SYTO 9) used is the original. The HRM reagent (Melt Dr, HRM reagent) included in the factory is 2x premix. During the process, it is not necessary to confirm the product by electrophoresis like HR-1, and the amplification curve can be obtained directly on the machine.

In addition, regarding the results of applying two kinds of fluorescent/nucleic acid dyes (LC green and SYBR green) to the HR-1 instrument: 1. Both kinds of fluorescence applied to the BCBghsp primer pair can successfully increase the target product, and Sample analysis is required using a glass capillary for HR-1. 2. Both need to go through HR-1 analysis can be performed after electrophoresis confirmation: in LC green, it can be added after the PCR reaction, which is more than the reagent amount, but LC green is pure fluorescence, and additional PCR premix is needed; in SYBR green, it is 2x premix There is no need to prepare additional PCR premix, but its shortcomings are because it contains SYBR green fluorescence, which cannot be confirmed by electrophoresis and is added. 3. Results of HR-1 analysis: 1. The Tm value (melting curve temperature) is similar between BC and Bg (LC green is 2.3 ° C, SYBR green is 2.34 ° C). 2. The value of Tm value in LC green is greater than the value of SYBR green (LC green BC is 89.73 ° C, Bg is 87.43 ° C; LC green BC is 87.56 ° C, Bg is 85.22 ° C), from which the results can be inferred SYBR green It is less able to detect the heteroduplex structure, and LC green can not only detect the heterogeneous double-strand structure, but the melting curve temperature can be observed to be wider (more detailed) than SYBR green, Liew et al. It is further pointed out that LC green can be used to rapidly detect heterologous double-stranded structures in homologous homologues without inhibiting the amplification of PCR products, without special probes or reagents, only specific primer pairs, one PCR and analysis. instrument. The application of LC green to HRM emphasizes that it can detect the difference of single base, which is often applied to single point mutation and drug resistance gene detection.

This experiment is to analyze the heat shock protein 70 of BC and Bg itself, using its multiple base differences, adding fluorescence and embedding it on the double-stranded nucleic acid, and then using the HRM principle to analyze the fluorescence degradation rate. Melting curve temperature of target gene, melting curve by BC and Bg Temperature difference The purpose of the distinction is sought, and the ultimate goal of the experiment is not to detect differences in single bases, so there is not much restriction on the use of fluorescent substances. In addition, in order to achieve quantitative purposes, the HRM analyzer (HR-1, Idaho technology) can only detect the fluorescence intensity and Tm value, and the quantitative effect cannot be achieved. However, the HRM experimental mode in the ViiA7 instrument can not only obtain the Tm. The value can also be used to determine the Ct value in the PCR system and further convert the nucleic acid concentration of the unknown worm.

Quantification of BC and Bg: The probe designed for 18S rDNA of BC and Bg did not reach the purpose of differentiation. Therefore, heat shock protein 70 was used for HRM test. The result is feasible and can be used in the HRM experimental mode of ViiA7. Successfully distinguishing between BC and Bg, the results of the Raw melt curve, the derivative melt curve, the aligned melt curves, and the amplification curve. The Ct value of the sample to be tested is obtained in an amplification plot, and the absolute quantitative conversion of the sample to be tested is performed using the positive control group of known concentration and its Ct value.

In summary, the high resolution melting method of the heat shock protein 70 of canine worm and Gibbs pine can successfully distinguish the two worms and use the known concentration positive control. The Ct value of the group is obtained to determine the concentration of the tested worm (up to an absolute quantitative value). The detection method is about four hours (including nucleic acid extraction and HRM on-board time), which is a very fast experimental method and greatly provides a clinical veterinarian clinic. Need to treat prime time. In the future, the technical content disclosed by the present invention can be utilized, which has the advantages of improving detection accuracy and reducing the occurrence of false positives. According to the technical contents, a rapid detection system for distinguishing canine size insects can be established, and a more efficient detection service can be provided. .

It can be seen from the above embodiments that the method for utilizing the gene detection and analysis character provided by the present invention has industrial utilization value, but the above description is only for explaining the preferred embodiment of the present invention, and those skilled in the art can Other various modifications are possible in light of the above teachings, but such changes are still within the spirit of the invention and the scope of the invention as defined below.

references

1. Birkenheuer, AJ, Levy, MG, Breitschwerdt, EB Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. Journal of Clinical Microbiology. 41(9) : 4172 ^~ 4177, 2003.

2. Lee, CC, Huang, CC, Tsang, CL, Chung, YT Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein (TRAP) gene of Babesia gibsoni isolates from dogs in Taiwan. Journal of veterinary medicine science. 72(10) : 1329 ^~ 1335, 2010.

3. Matjila, PT, Leisewitz, AL, Jongejan, F., Bertschinger, HJ, PenZhorn, BL Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Afica. Veterinary Parasitology. 157: 123 ^~ 127, 2008.

4. Zahler, M., Schein, E., Rinder, H., Gothe, R. Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs. Parasitology Research. 84: 544 ^~ 548, 1998.

5. Wittwer, CT, Reed, GH, Gundry, CN, Vandersteen, JG, Pryor, RJ High-resolution genotyping by amplicon melting analysis using LC Green. Clinical Chemistry. 49(6): 853 ^~ 860, 2003.

6. Liew, M., Pryor, R., Palais, R., Meadows, C., Erali, M., Lyon, E., Wittwer, C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons . Clinical Chemistry. 50(7): 1156 ^~ 1164, 2004.

Figure 1 is a schematic diagram showing the results of 18S rDNA sequence alignment analysis of Babesia canis (BC) and Babesia gibsoni (Bg).

2 is a schematic diagram of a nucleic acid extraction by a clinical blood sample using its blood cell layer (RBC/buffy coat).

Figure 3 is a schematic diagram of the 18S rDNA primer pair (BCBg308F/R) of Babesia canis (BC) and Babesia gibsoni (Bg).

Fig. 4 is a schematic diagram showing the results of electrophoresis of a BCBg308F/R by a 18S rDNA primer combined with BC and Bg.

Fig. 5 is a schematic diagram showing the introduction of nested PCR primers for the TLP sequence of Bg (the green frame is a 1st PCR primer pair and the blue frame is a 2nd PCR primer pair).

Figure 6 is a schematic diagram showing the results of one PCR performed on the nested PCR of the BgTRAP1091 primer pair.

Figure 7 is a schematic diagram showing the results of secondary PCR of nested PCR of BgTRAP481 primer pair.

Figure 8 is a schematic diagram of the BC and Bg probes designed with the primer pair BCBg308F/R (blue frame for BC probe1 and red for Bg probe2).

Figure 9 is a schematic diagram of an amplification plot of the BC probe 1 test of BC and Bg samples in the ViiA7 "standard curve" experimental mode.

Figure 10 is a schematic diagram of an amplification plot of the BC probe 2 test of BC and Bg samples in the ViiA7 "standard curve" experimental mode.

Figure 11 is a schematic diagram showing the position of restriction enzyme digestion by Mfe I (which must act on BC and Bg at the blue frame and only Bg at the red frame).

Fig. 12 is a schematic diagram showing the results of restriction enzyme digestion of products of BC and Bg samples by PCR amplification of BCBg308F/R by BC and Bg co-introduction.

Figure 13 is a schematic representation of the position of the primer pair (green box) of the heat shock protein 70 sequence of BC and Bg, wherein the blue box shows a schematic representation of the difference between BC and Bg.

Fig. 14 is a schematic diagram showing the results of PCR performed by heat shock protein 70 on BC and Bg samples.

Fig. 15 is a view showing the results of thermal dissolution curve analysis of BC and Bg samples by PCR amplification products of heat shock protein 70.

Figure 16 is a schematic diagram of the Taq test results.

Figure 17 is a schematic diagram showing the results of PCR confirmation before HR-1 analysis.

Figure 18 is a graph showing the results of a derivative plot of the PCR product of Figure 17 after HR-1 analysis.

Figure 19 shows the results of PCR confirmation of BC and Bg samples before HR-1 analysis (without LCgreen)

Figure 20 is a schematic diagram showing the results of the HRM derivative plot of BC, Bg heat shock protein 70 (Fig. 19 product + LCgreen).

Fig. 21 is a schematic diagram showing the results of PCR confirmation (SYBR green premix) of BC and Bg samples before HR-1 analysis.

Fig. 22 is a schematic diagram showing the results of the HRM derivative plot of BC, Bg heat shock protein 70 (Fig. 21 product + LCgreen).

Fig. 23 is a schematic diagram showing the results of 18S rDNA amplification of BC and Bg samples by BCBgprobe2 primer pair (the results of PCR confirmation before HR-1 analysis).

Fig. 24 is a schematic diagram showing the results of the derivative plot of HRM of BC, Bg 18S rDNA with HR-1 (Fig. 23 product + LCgreen).

Figure 25 is a schematic illustration of the BC heat shock protein 70 amplification plot in ViiA7 "High Resoultion Melting" experimental mode.

Figure 26 is a schematic diagram showing the heat shock protein 70 amplification plot of Bg in the "High Resoultion Melting" (HRM) experimental mode of ViiA7.

Figure 27 is a graphical representation of the Raw melt curve for BC, Bg, and BCBg mixed heat shock protein 70 with ViiA7.

Figure 28 is a graph showing the results of a derivative melt curve of HRM by VII, Bg and BCBg mixed heat shock protein 70 with ViiA7.

Fig. 29 is a graph showing the results of normalization of HRM signals by ViiA7 using BC, Bg and BCBg mixed heat shock protein 70.

Figure 30 shows BC, Bg and BCBg mixed heat shock protein 70 with ViiA7 Schematic diagram of the difference plot of HRM.

Figure 31-1 is a schematic diagram of semi-dilute dilution using a known concentration of pBC and pBg (10 ug/uL) in a 1:1 mix.

Figure 31-2 shows the difference between the semi-sequence dilution (seim-dilute) using a known 1:1 pBC and pBg concentration (10 ug/uL) and the HRM with ViiA7. BC+ is known. A schematic representation of a positive specimen.

Figure 32 is a derivative melt curve for the HRM with ViiA7 using a known 1:1 mixture of pBC and pBg concentrations (10 ug/uL) in a 1:1 mixture, BC+ is known. A schematic representation of a positive specimen.

Figure 33 is a schematic diagram showing the results of the sequence of Babesia canis and NCBI blast results.

Figure 34 is a schematic diagram showing the results of sequencing of Babesia gibsoni and NCBI blast results.

FIG 35 is a Jibu Song Babesia (Babesia gibsoni) sequencing schematic NCBI blast results and results TRAP gene.

Figure 36 shows the results of sequencing of Babesia canis heat shock protein 70 and NCBI blast results.

Figure 37 is a schematic diagram showing the results of sequencing of heat shock protein 70 of Babesia gibsoni and NCBI blast results.

<110> Kelong Meikes Biotechnology Co., Ltd.

<120> Method for distinguishing the classification of canine worms

<130> 101B0405

<160> 4

<170> Pai Printing Version 3.5 (PatentIn version 3.5)

<210> 1

<211> 1944

<212> DNA

<213> Babesia canis

<220>

<221> gene

<222> (1)..(1944)

<400> 1

<210> 2

<211> 1941

<212> DNA

<213> Gibson gibsoni ( Babesia gibsoni )

<220>

<221> gene

<222> (1)..(1941)

<400> 2

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> None

<220>

<221> Other features

<222> (1)..(18)

<223> Introduction BCBghsp231F

<400> 3

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> None

<220>

<221> Other features

<222> (1)..(20)

<223> Introduction BCBghsp231R;r=g or a;y=t/u or c;k=g or t/u

<400> 4

Claims (5)

A method for distinguishing a type of canine phobia, the steps comprising: (a) providing a nucleic acid sample; (b) performing a quantity of real-time polymerase chain reaction (Q real-time PCR) on the nucleic acid sample; Performing a melting curve analysis to distinguish the canine worm typing of the nucleic acid sample; wherein the canine genus classification includes Babesia canis (BC) and Gibbs pine worm ( Babesia) Gibsoni , Bg), and the primer used in the real-time quantitative polymerase chain reaction pair SEQ ID NO: 3 and SEQ ID NO: 4 for encoding the heat shock protein 70 in the nucleic acid sample SEQ ID NO: 1 and SEQ ID NO: 2 segments of the gene. The method of claim 1, wherein the step (c) further comprises performing a High-Resolution Melting (HRM) analysis. The method of claim 1, wherein the nucleic acid sample is extracted from a blood sample of a body. The method of claim 3, wherein the blood sample is a blood cell sample after separation by blood cell plasma. The method of claim 1, wherein the step (C) further comprises using an LC green dye, a SYBR green dye or a SYTO 9 dye.