KR102027120B1 - Method for detecting Lysinibacillus fusiformis, a suspicious pathogen of Bombus terrestris using ultra-rapid Polymerase Chain Reaction technique - Google Patents

Abstract

Lysinibacillus suspected pathogen for suspicion of Western dune using ultrafast PCR A method for detecting of fusiformis is disclosed . According to a detection method according to an embodiment of the present invention, the presence of Lysinibacillus fusiformis is detected by performing an ultrafast PCR process with a primer composition comprising primer pairs of SEQ ID NOs: 1 and 2. In particular, Lysinibacillus fusiformis to optimize the conditions for specific genes high-speed PCR process, to minimize the time it takes to Lysinibacillus fusiformis detected by Western common carder bee adult suspected to be infected with Lysinibacillus fusiformis. By applying such optimized Lysinibacillus fusiformis- specific ultrafast PCR, infection of Lysinibacillus fusiformis can be determined in 4 minutes and 22 seconds based on the number of 1.0 × 0 ⁸ molecules, and quantitatively measuring recombinant DNA of at least 1.0 × 0 ¹ molecule. can do. In addition, detection of pathogens using suspicion of infection caused by Lysinibacillus fusiformis- specific gene ultrafast PCR showed a positive response, which is useful for quantitative detection in the field as well as in the laboratory.

Description

Method for detecting Lysinibacillus fusiformis, a suspicious pathogen of Bombus terrestris using ultra-rapid Polymerase Chain Reaction technique}

The present invention relates to a technique for rapidly detecting Lysinibacillus fusiformis, which is one of the suspected pathogens of Bombus terrestris using ultra-fast PCR (Polymerase Chain Reaction) method.

The use of pollen- mediated insects has a great influence on the development of plant vegetable farming methods. Bees ( Apis mellifera ) and Western back bees are used as the main pollen insects. The research on the practical use of Western Back Stars has been actively made and commercialized in Europe, the United States, and Canada. In the early days, Western Back Young Bees were used for pollen mediation. In Korea, as the area of facility cultivation increased, Western duvets of 2,300 Bonggun were first imported in 1994, and more than 30,000 Bonggun were imported in 2004. However, afterwards, domestic production of Western bumblebees has increased significantly, especially since 2010, which is seeking to export extra Western bumblebees, exceeding domestic demand.

Domestic studies on the pathogens possessed by Western bumblebees confirmed that the honeybee virus, DWV (Deformed Wing Virus), also existed in western bumblebees and bumblebees, followed by seven types of honeybee viruses, namely DWV and BQCV. (Black Queen Cell Virus), IAPV (Israeli Acute Paralysis Virus), CWV (Cloudy Wing Virus), CBPV (Chronic Bee Paralysis Virus), SBV (SacBrood Virus), KBV (Kashmir Bee Virus) were confirmed to exist. In addition, Lysinibacillus fusiformis and Klebsiella oxytoca ) were isolated from Western bumblebees, suggesting that they may be pathogenic bacteria of Western bumblebees.

However, as trade between countries is active, biological / environmental issues such as the extinction of indigenous species due to the influx of invasive species, as well as the infection by bacteria or pathogens introduced with the invasive species are highlighted. However, to date, no detection method has been proposed to test pathogens at the same time, and quarantine standards have been established at home and abroad in the recent export promotion of domestic western dune bees. Therefore, in the context of active import and export of Western back horned bee, the demand for the establishment of technology and quarantine criteria for screening pathogens for western back bleeding is increasing and is critical.

"Isolation of Pathogenic Bacteria for Bombus terrestris and Bignites", Won-Tae Kim et al. 8, Journal of The Korean Beekeeping Society, Vol. 23, No. 1, 2008, pp. 13-20 "Development of Real-Time Polymerase Detection Method for 11 Duyoung Beetle Pathogens", Min Sang-hyun et al. 5, Journal of The Korean Beekeeping Society, Vol. 32, No. 2, 2017, pp. 99-109

One problem to be solved by the present invention is to provide a method for detecting the presence or absence of Lysinibacillus fusiformis , one of the suspected pathogens of the Western dung bee .

Another problem to be solved by the present invention is Lysinibacillus , which is one of the suspected pathogens of Western dune bees By developing a primer set for the detection of fusiformis , Lysinibacillus in Western dune bees using ultrafast PCR It provides a way to quickly and accurately determine whether fusiformis exists.

According to the detection method of the suspicion of late bumblebee pathogen Lysinibacillus fusiformis using the ultrafast PCR according to an embodiment of the present invention for solving the above problems, the ultrafast PCR process using a primer composition comprising a primer pair consisting of SEQ ID NO: 1 and 2 The presence of suspected pathogen, Lysinibacillus fusiformis , is detected by the following procedure.

According to one aspect of the embodiment, the annealing step in the ultra-fast PCR process may be performed at 57 ℃.

According to another aspect of the embodiment, the primer pair of SEQ ID NO: 1 and 2 in the ultrafast PCR process may have a concentration of 4μM.

According to the detection method of the suspicion of suspected late bumblebee pathogen Lysinibacillus fusiformis using the ultra-fast PCR method according to the embodiment of the present invention, it is possible to detect the presence of Lysinibacillus fusiformis quickly and accurately. In particular, Lysinibacillus fusiformis to optimize the conditions for specific genes high-speed PCR process, to minimize the time it takes to Lysinibacillus fusiformis detected by Western common carder bee adult suspected to be infected with Lysinibacillus fusiformis. By applying such optimized Lysinibacillus fusiformis- specific ultrafast PCR, infection of Lysinibacillus fusiformis can be determined in 4 minutes and 22 seconds based on the number of 1.0 × 10 ⁸ molecules, and quantitatively measuring recombinant DNA of at least 1.0 × 10 ¹ molecules. can do. Also, Lysinibacillus Fusiformis- specific genes were found to be positive for the detection of pathogens using suspicion of infection by using ultrafast PCR, which is useful for quantitative detection in the field as well as in the laboratory.

1 is a graph showing the experimental results for determining the optimal hybrid temperature in Lysinibacillus fusiformis specific ultrafast PCR.
Figure 2 is a graph showing the experimental results for determining the optimal concentration of specific primers in Lysinibacillus fusiformis specific ultrafast PCR.
Figure 3 shows the experimental results of obtaining the minimum time of the hybridization and polymerization step to set the minimum detection time in Lysinibacillus fusiformis specific ultrafast PCR.
Figure 4 is a graph showing the fluorescence value when the hybridization time is shortened in Lysinibacillus fusiformis specific ultrafast PCR.
5 is a graph showing the results of experiments to determine the minimum detection limit of Lysinibacillus fusiformis specific genes.
FIG. 6 is a diagram illustrating a process of obtaining a regression equation based on the relationship between the number of Lysinibacillus fusiformis molecules and the critical cycle (C _T ) applied to Lysinibacillus fusiformis specific ultrafast PCR.
Figure 7 is a graph showing the results of experiments to determine the optimal amount of leprosy nucleic acid nucleic acid for the detection of Lysinibacillus fusiformis- specific genes.
8 is a view showing the results of experiments performed Lysinibacillus fusiformis specific ultra-fast PCR by collecting the Western dung bee sold in Korea.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Terms and words used herein are terms selected in consideration of functions in the embodiments, and the meaning of the terms may vary according to the intention or custom of the invention. Therefore, the terminology used in the embodiments to be described later, according to the definition when specifically defined in the present specification, if there is no specific definition should be interpreted as meaning generally recognized by those skilled in the art.

The inventors of the present application have confirmed that when performing ultrafast PCR with the primer set including the primer sets including the primer sets of SEQ ID NOs: 1 and 2, which will be described later, it is possible to quickly and accurately detect the suspected leprosy suspected pathogen Lysinibacillus fusiformis . It came to invention. In addition, the inventors of the present application conducted experiments to obtain optimal process conditions for detecting Lysinibacillus fusiformis quickly and accurately using the Lysinibacillus fusiformis specific ultrafast PCR process. by applying a specific high-speed Lysinibacillus fusiformis PCR process through the body of the common carder bee it was detected Lysinibacillus fusiformis.

In the following the design and manufacture a primer set that can be used to detect the Western common carder bee suspected pathogen Lysinibacillus fusiformis a high-speed PCR method, and by using this process of determining the optimal process conditions for detecting the Western common carder bee suspected pathogen Lysinibacillus fusiformis, as well as through them Lysibacillus suspected pathogen, through real samples The detection result of fusiformis will be described in detail.

[Materials and methods]

Sample acquisition and separation of DNA (DeoxyriboNucleic Acid) and RNA (RiboNucleic Acid)

Common carder bee samples were used 8 March 2016 by Western common carder bee (Bombus terrestris) obtained from the light article Gyeonggi, Jeolla Province Boseong, Gyeongsangbuk-do Cheongsong, Gyeongsangnam-do Miryang, Gyeongsangnam-do Changwon.

In order to isolate genomic DNA (GDNA) of Duyoung Bee, first, the obtained sample was crushed by a known method, and then the method of G-spin ^TM Total DNA Extraction Kit (iNtRON, Korea) was followed. And total RNA isolation was performed according to the manufacturer's instructions using the R & A Blue Kit (Takara, Korea). The extracted back-young bee gDNA and total RNA were quantified using a spectrophotometer, stored at -70 ° C and used for subsequent experiments.

Backyoung bee pathogen Lysinibacillus fusiformis Specific primers and secured recombinant plasmid DNA

After confirming sequencing information in GenBank (NCBI) for the pathogen, a pair of specific primers were designed. At this time, the length of the PCR product was designed to be between 120 base pairs (bp) to 250 bp to be suitable for quantitative ultrafast PCR. Lysinibacillus fusiformis was obtained from ATCC 7055, a standard monarch, from the Korean Collection for Types Cultures, extracted genomic DNA (gDNA), PCR amplified most of the Glutamyl-tRNA amidotransferase gene, and cloned into pBX-vector. It was used as a standard substrate.

Table 1 discloses a pair of Lysinibacillus fusiformis specific primers and a pair of oligonucleotides for the amplification of Lysinibacillus fusiformis -specific primers. Lysinibacillus fusiformis specific primer pairs or recombinant DNA as shown in Table 1 were named pLF-159, which carries 4568067-4568195 bases (159 bases in length) of GenBank CP010820.1 ( Lysinibacillus fusiformis full genome) Doing. That is, the target DNA Glutamyl tRNA amidotransferase has 4568067-4568195 bases (length 159 bases) and is located in GenBank CP010820.1.

Lysinibacillus fusiformis  Optimization of PCR Conditions for Ultrafast Detection of Specific Genes

Condition optimization of the ultrafast PCR process was performed as follows. First, pLF-159, a recombinant DNA, was used as a template for ultrafast PCR, quantified by OD260 (Optical Density 260nm), and used for ultrafast PCR based on 1.0 × 10 ⁶ molecules. Ultrafast PCR instrument was used GENECHECKER ^TM (Genesystem Co., Korea). The temperature and time conditions of the ultrafast PCR process were based on 15 seconds of 95 ° C initial denaturation, 3 seconds of 95 ° C denaturation, 3 seconds of hybridization, and 3 seconds of 72 ° C elongation. Optimum annealing (annealing) temperature was found at 55-65 ℃ range, optimum primer concentration was each cycle threshold (Threshold Cycles, C _T) value and measuring the maximum fluorescence intensity in 4μM, 2μM, 1μM, 0.5μM, 0.25μM It was. At this time, the temperature and time conditions of the ultrafast PCR were readjusted based on the C _T value and the maximum fluorescence value from the above standard conditions.

Lysinibacillus fusiformis  Detection limit measurement according to minimum detection time of specific gene

In order to determine the limit of specific detection at the minimum detection time of Lysinibacillus fusiformis specific gene, melting point temperature analysis of amplified products was performed after real-time PCR reaction. The melting point temperature analysis was also shown in the graph by measuring the fluorescence value in increments of 1 ° C. from 60 ° C. to 90 ° C. at 1 second intervals. The graph was micronized to identify the peak of temperature change. This peak is synonymous with the temperature of midpoint (Tm) and is given in Tm for the PCR product. Non-specific reaction products could be distinguished based on Tm values of PCR products for standard substrates. Lysinibacillus fusiformis reduced from 3 seconds for each of the rotation-modified, mixed, the time of the polymerization of high-speed PCR, respectively to measure the limits of specificity unique in the minimum sensing time of high-speed PCR detection to 1 second for each time and temperature conditions C _T The time at which the value and the C _T value were measured, ie the critical cycle time (C _T time), was measured. Recombinant plasmid DNA for pathogen specific genes was precisely measured at an absorbance of 260 nm with a spectrophotometer to calculate the number of standard substrate molecules in each DNA solution. Each recombinant plasmid DNA was serially diluted 10-fold from 1 × 10 ⁸ molecules to 1 × 10 ⁰ molecules to use template DNA in each PCR.

Lysinibacillus fusiformis  Amount and Standard Assay of Optimal Dominimal Bovine Nucleic Acids for Detection of Specific Genes

Lysinibacillus To determine the amount of nucleic acid suitable for the detection of fusiformis- specific genes, Lysinibacillus The amplification amount of Lysinibacillus fusiformis- specific gene was confirmed using the nucleic acid extracted from fusiformis as a template. The PCR reaction was performed by 5 μl of 2 × api master mix and 1 μl of L. fusiformis detection F2 / R2 (final concentration of 4 μM) primers followed by a doubly-depleted nucleic acid (up to 50 ng to 1 ng dilution) in total of 10 μl. As a positive control, recombinant DNA containing 1.0 × 10 ⁶ copies of Lysinibacillus fusiformis gene was used to compare the synthesis of Lysinibacillus fusiformis PCR products with specific median temperature (Tm). The time and temperature conditions for each step of the ultrafast PCR are based on the conditions optimized in the previous experiments. After 15 seconds of 95 ° C initial pre-denaturation, 1 second of 95 ° C denaturation, 2 seconds of hybridization, and 2 seconds of 72 ° C extension are standard. And a total of 50 revolutions were performed.

In the domestic western back bee Lysinibacillus fusiformis  Detection using specific ultrafast PCR

In order to determine the pathogen infection of Western dune bee samples in Korea, gDNA and cDNA were used for each western dune bee pathogen in each of 50 western dune bees produced in Suwon, Jeolla Province, Boseong, Cheongsong, Gyeongsangbuk-do, and Changwon, Gyeongsangnam-do. Ultrafast PCR was performed for Lysinibacillus fusiformis . The PCR reaction was performed by 5 μl of 2 × api master mix and 1 μl of L. fusiformis detection F2 / R2 (final concentration 4 μM) in a total of 10 μl. Lysinibacillus The fusiformis specific ultra-fast PCR was performed using GENECHECKER ^TM (Genesystem Co., Korea), and PCR conditions were 15 seconds after 95 ° C initial denaturation (pre-denaturation), 95 ° C for 1 second denaturation, and 57 ° C for 2 seconds hybridization. ), A total of 50 rotations were carried out under the condition of 72 ℃ 2 seconds extension (extension).

[Results and Discussion]

Lysinibacillus fusiformis  Optimization of PCR Conditions for Ultrafast Detection of Specific Genes

In order to determine the optimal hybridization temperature in Lysinibacillus fusiformis specific ultrafast PCR, pLF-159 of recombinant DNA 1.0 × 10 ⁶ molecules was used, and the temperature and time conditions of ultrafast PCR were 15 seconds after 95 ° C initial denaturation optimized in the previous experiment, 95 A total of 50 rotations were performed based on 3 ° C. denaturation 3 seconds, 55-65 ° C. hybrid 3 seconds, and 72 ° C. elongation 3 seconds, and the experimental results are shown in a graph in FIG. 1.

1 is a graph showing the experimental results for determining the optimal hybrid temperature in Lysinibacillus fusiformis specific ultrafast PCR. As described above, the experimental result of FIG. 1 is a hybrid step of ultrafast PCR in a temperature range of 55 ° C to 65 ° C, and the remaining process steps constituting the ultrafast PCR are the same as described above. Referring to FIG. 1, the fastest critical cycle (C _T ) was measured at 23.28 cycles at a hybrid temperature of 57 ° C., and the PCR run time, that is, the critical cycle time (C _T time), was 10 minutes 44 seconds. . Therefore, the optimal hybridization temperature in Lysinibacillus fusiformis specific ultrafast PCR was determined to be 57 ° C, which showed excellent results in both C _T (23.28 cycles) and final fluorescence (F = 221).

In addition, in order to obtain the optimal concentration of Lysinibacillus fusiformis- specific primers in Lysinibacillus fusiformis- specific ultrafast PCR, experiments were performed using primers at concentrations of 4 μM, 2 μM, 1 μM, 0.5 μM, and 0.25 μM, respectively, and the results are shown in FIG. 2. .

Figure 2 is a graph showing the experimental results for determining the optimal concentration of specific primers in Lysinibacillus fusiformis specific ultrafast PCR. In determining the optimal concentration of specific primers based on the experimental results of FIG. 2, the fastest critical cycle (C _T ) value and the final fluorescence value were used as a reference. Referring to FIG. 2, the ultrafast PCR at the final concentration of 4 μM showed the fastest critical cycle (C _T ) value of 20.28 cycles, and the final fluorescence value was also determined to be the highest. Therefore, the optimal primer concentration in Lysinibacillus fusiformis specific ultrafast PCR showed the fastest detection time at the critical cycle (C _T ) value (20.28 ± 0.08 cycles) and the critical cycle time (C _T time, 7 minutes 56 seconds), and the final fluorescence. It was determined to be 4 μM with the best results in all values (223 ± 17.67).

Lysinibacillus fusiformis  Detection limit according to minimum detection time of specific gene

In the ultrafast PCR using 1.0 × 10 ³ pLF-159 molecules as substrates, the hybrid cycle time of each rotation was adjusted to 3 seconds. As a result, the critical cycle (C _T ) value was reduced to 28.77 cycles, and the critical cycle time (C _T time) was In 9 minutes and 40 seconds, amplification of Lysinibacillus fusiformis- specific genes was confirmed. High-speed PCR reducing each hybrid time only 2 seconds, 1 second at the same conditions showed a result that the value that the threshold cycle (C _T) increases the hybrid time The shorter quality, the threshold cycle time (C _T time) is rather The increase and decrease was observed, but the reduction was apparent when the hybrid phase was given to 2 seconds.

Figure 3 shows the experimental results of obtaining the minimum time of the hybridization and polymerization step to set the minimum detection time in Lysinibacillus fusiformis specific ultrafast PCR. In the experiment of FIG. 3, in each cycle of PCR, the denaturation step was fixed to 1 second, and the hybridization and polymerization steps were reduced from 3 seconds to 2 seconds and 1 second, respectively. Referring to FIG. 3, the polymerization time was also reduced to 3 seconds, 2 seconds, and 1 second in the PCR reaction. As a result, the critical cycle time (C _T time) was 8 minutes and 29 seconds in the ultrafast PCR which gave the polymerization time of 1 second. Compared with 3 seconds, it can be seen that a significant decrease of more than 1 minute was measured. According to this, it can be seen that specific amplification is possible even when each denaturation, hybridization, and polymerization time of Lysinibacillus fusiformis specific ultrafast PCR is set to 1 second.

Figure 4 is a graph showing the fluorescence value when the hybridization time is shortened in Lysinibacillus fusiformis specific ultrafast PCR. Referring to FIG. 4, when the denaturation time, hybridization time, and polymerization time are 1 second, 2 seconds, and 1 second, respectively, the fluorescence curve indicating DNA amplification shows a relatively normal increase according to the initial template amount, but the degeneration time, When the hybridization time is shortened to the hybridization time and the polymerization time of 1 second, 1 second, and 1 second, respectively, the shortest time condition results, the fluorescence curve indicating DNA amplification is greatly distorted in the amplification of the initial template DNA of 1 × 10 ³ molecules or less. This can be seen frequently. According to this, it turns out that quantitative detection becomes difficult with reduction of hybridization time.

Therefore, Lysinibacillus fusiformis specific ultrafast PCR was performed for 1 second, 2 seconds, and 2 seconds for each rotation denaturation time, hybrid time, and polymerization time in consideration of the stability of the PCR system.

In addition, the minimum detection time for specific amplification of Lysinibacillus fusiformis gene was measured by Lysinibacillus fusiformis specific ultrafast PCR. 1.0 × 10 ⁸ pLF-159 molecule was used as a template and optimized Lysinibacillus With the composition of fusiformis specific ultrafast PCR, the denaturation time, hybridization time, and polymerization time were set to 1 second, 2 seconds, and 2 seconds under optimized temperature conditions, and 50 cycles were performed. After 15 seconds of initial denaturation, the critical cycle line (C _T line) was reached in only 13.59 cycles, and the critical cycle time (C _T time) was 4 minutes and 8 seconds. The maximum fluorescence value was shown before and after 38 cycles (C _T time 11 minutes 34 seconds) and 50 cycles of ultrafast PCR were completed at 16 minutes 47 seconds.

Lysinibacillus fusiformis  Detection Limits and Quantitative Ranges for Specific Ultrafast PCR

5 is a graph showing the results of experiments to determine the minimum detection limit of Lysinibacillus fusiformis specific genes. In the graph shown in Figure 5 is a Lysinibacillus fusiformis specific ultra-fast PCR reactions described above, the PCR reaction is performed by setting the above-mentioned stable time conditions, that is, denaturation time, hybrid time, polymerization time in each cycle 1 second, 2 seconds, 2 seconds, Lysinibacillus Fusiformis specific DNA recombinant DNA, starting with 1.0 × 10 ⁸ , was serially diluted to 1.0 × 10 ⁰ molecules and used as a template for ultra-fast PCR.

Referring to Figure 5, Lysinibacillus under the reaction conditions The fusiformis gene can be successfully amplified even in the initial template of 1.0 × 10 ¹ molecule, indicating excellent sensitivity. And when the difference in the critical cycle (C _T ) value between the initial molecular weight reduced by 10 ⁻¹ is dCT, the average dCT value from 1.0 × 10 ⁸ molecules to 1.0 × 10 ¹ molecule is about 3.30 cycles (see Fig. 6) In the initial template of 1.0 × 10 ¹ molecule, the average critical cycle (C _T ) value was 36.74 cycles, the average critical cycle time (C _T time) was 11 minutes 11 seconds, and the center temperature (Tm) value was 78.47 ° C. It was included in the effective range of Lysinibacillus fusiformis , that is, the measured midpoint temperature (Tm) range of all PCR products, 78.55 ± 0.73 ° C. Therefore, it is judged that the amount of template required for stable detection is 1.0 × 10 ¹ molecule or more.

In addition, the Lysinibacillus fusiformis- specific ultrafast PCR (indicated by N in FIG. 5) in which distilled water was added instead of the Lysinibacillus fusiformis- specific gene showed amplification above the critical cycle line (C _T line) at about 40 cycles. The value is measured at 74.56 ° C., which shows a difference of 3.99 ° C. from the midpoint temperature (Tm) of the Lysinibacillus fusiformis effective range, making it easy to confirm that this is not specific amplification.

FIG. 6 is a diagram illustrating a process of obtaining a regression equation based on the relationship between the number of Lysinibacillus fusiformis molecules and the critical cycle (C _T ) applied to Lysinibacillus fusiformis specific ultrafast PCR. In each of the experimental results in FIG. 6, each critical cycle (C _T ) value was measured according to the amount of template DNA serially diluted in 10 ⁻¹ units in ultrafast PCR using pLF-159, a recombinant DNA. Referring to FIG. 6, the regression equation according to the experimental result is y = -3.1871x + 39.192 (y = numerator of initial Lysinibacillus fusiformis ), where the regression coefficient is R ² = 0.9947. In addition, it can be seen that the quantitative detection is possible through the obtained regression equation, and the quantitative range for accurate quantification is that the template DNA is 1.0 × 10 ¹ molecule or more to 1.0 × 10 ⁸ molecules.

Lysinibacillus fusiformis  Amount and Standard Assay of Optimal Dominimal Bovine Nucleic Acids for Detection of Specific Genes

Figure 7 is a graph showing the results of experiments to determine the optimal amount of leprosy nucleic acid nucleic acid for the detection of Lysinibacillus fusiformis- specific genes. In the experiment for obtaining the graph of FIG. 7, genomic DNA (gDNA) was extracted from an average of 7.27 µg from one adult adult H. pylori in order to establish a nucleic acid range of H. pylori suitable for Lysinibacillus fusiformis specific ultrafast PCR. For nucleic acid, 50ng, 25ng, 10ng, and 1ng, respectively, were used to detect Lysinibacillus fusiformis gene.

Referring to FIG. 7, it can be seen that an amplification curve is formed on the Lysinibacillus fusiformis gene amplification fluorescence graph, and the highest final fluorescence value is shown at 25 ng as a template and the lowest fluorescence value at 10 ng. The critical cycle (C _T ) value of Lysinibacillus fusiformis was measured at 50ng to 20.93 cycles (critical cycle time (C _T time = 6 min 22 sec), and then at 25ng to 23.45 cycles (critical cycle). time (C _T time) = 7 minutes 8 seconds), 26.64 cycle at 10ng (threshold cycle time (C _T time) = 8 minutes 7 seconds), 28.18 cycle at 1ng (threshold cycle time (C _T time) = 8 minutes 35 Seconds) were measured. Thus, based on the fluorescence graph, the final fluorescence value, and the measured critical cycle (C _T ) value and the critical cycle time (C _T time), the optimal amount of dorsal bee nucleic acid was determined to be 50ng for PCR amplification.

Lysinibacillus fusiformis  Suspicion of infection Lysinibacillus fusiformis  Detection by specific ultrafast PCR

In order to evaluate the effectiveness of Lysinibacillus fusiformis specific ultrafast PCR according to an embodiment of the present invention, Western dune bees were sold in Korea and Lysinibacillus fusiformis specific ultrafast PCR was performed. More specifically, gDNA was isolated from Western dune beacons suspected of Lysinibacillus fusiformis infection among western dune bee samples sold in Suwon, Boseong, Changwon, and Cheongsong, respectively. Lysinibacillus fusiformis specific ultrafast PCR was performed using 50ng. The ultra fast PCR process conditions were 15 seconds after the initial degeneration of 95 ° C, a total of 50 rotations were performed based on 95 ° C modification of 1 second, 57 ° C hybridization of 2 seconds, and 72 ° C extension of 2 seconds, and the experimental results are shown in FIG. 8. have. Referring to FIG. 8, Lysinibacillus fusiformis gene was detected in each of the samples used in Western dune bee, that is, Suwon, Boseong, Changwon, and Cheongsong, respectively. In particular, the sample of Changwon was 1,000 times higher than the other three samples. Many Lysinibacillus fusiformis genes were confirmed to exist.

Table 2 shows the critical cycle (C _T ) and midpoint temperature (Tm) values for each of the products of the Lysinibacillus fusiformis specific ultrafast PCR. Referring to Table 2, Lysinibacillus fusiformis- specific ultrafast PCR showed that amplification of Lysinibacillus fusiformis- specific genes was observed in all samples, and the melting point analysis of each product showed that the amplification of Lysinibacillus fusiformis- specific genes could be confirmed by the center temperature (Tm) value. . In addition, the measured Tm values of Lysinibacillus fusiformis were 78.47 ℃, 77.82 ℃, 78.47 ℃, and 78.47 ℃, respectively, in Suwon, Boseong, Changwon, and Cheongsong, and they were within the effective range of positive amplification of Lysinibacillus fusiformis (78.55 ± 0.73 ℃). It can be determined by amplification of Lysinibacillus fusiformis gene.

These results show for the first time that Lysinibacillus fusiformis is actually present in the carcasses of Western dupe bees that are sold in Korea. Lysinibacillus fusiformis was infected with western dupe bees as an initial infectious bacterium. It would be difficult to determine whether it was simply grown in the body. The amount of Lysinibacillus fusiformis contained in each body was 1.4 × 10 ⁴ molecules / horse, 8.8 × 10 ² molecules / horse, 2.2 × 10 ⁷ molecules / horse, 7.6 × 10 ³ molecules / water in the order of Suwon, Boseong, Changwon, Cheongsong. It was a small number, which I think is likely to act as an infectious agent.

As described above, the detection method according to an embodiment of the present invention detects the presence of Lysinibacillus fusiformis by performing an ultrafast PCR process with a primer composition comprising a primer pair of SEQ ID NO: 1 and 2. In particular, Lysinibacillus fusiformis to optimize the conditions for specific genes high-speed PCR process, to minimize the time it takes to Lysinibacillus fusiformis detected by Western common carder bee adult suspected to be infected with Lysinibacillus fusiformis. By applying such optimized Lysinibacillus fusiformis- specific ultrafast PCR, infection of Lysinibacillus fusiformis can be determined in 4 minutes and 22 seconds based on the number of 1.0 × 10 ⁸ molecules, and quantitatively measuring recombinant DNA of at least 1.0 × 10 ¹ molecules. can do. Also, Lysinibacillus Fusiformis- specific genes were found to be positive for the detection of pathogens using suspicion of infection by using ultrafast PCR, which is useful for quantitative detection in the field as well as in the laboratory.

Although the present invention has been described in detail with reference to preferred embodiments, the present invention is not limited to the above-described embodiments, and various modifications may be made by those skilled in the art within the scope of the technical idea of the present invention. It is possible.

Claims (3)

Method for detecting the presence of suspicion of pathogens suspected Lysibacillus fusiformis by performing an ultra-fast PCR process with a primer composition comprising a primer pair of SEQ ID NO: 1 and 2.
The method of claim 1,
The annealing step in the ultra-fast PCR process is characterized in that it is carried out at 57 ℃.
The method according to claim 1 or 2,
The primer pair of SEQ ID NO: 1 and 2 in the ultra-fast PCR process, characterized in that having a concentration of 4μM.

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