KR20240177796A - LAMP primer set for specifically detecting Mycobacterium avium ssp paratuberculosis and use thereof - Google Patents

Abstract

The present invention relates to a LAMP primer set for specifically detecting Johne's disease and its use. The ISMap02 or IS1311 LAMP primer set for detecting Johne's disease of the present invention specifically detects only Johne's disease and exhibits improved sensitivity compared to existing diagnostic methods. In addition, since the LAMP method does not require specialized personnel, it can be easily used in the field to diagnose Johne's disease.

Description

{LAMP primer set for specifically detecting Mycobacterium avium ssp paratuberculosis and use thereof}

The present invention relates to a LAMP primer set for specifically detecting Yonephritis and its use.

Johne's Disease (Bovine paratuberculosis) is a chronic digestive disease that occurs in ruminants such as cattle, sheep, goats, and deer. It is distributed all over the world including the United States, and in Korea, there have been cases of Johne's disease in Korean cattle, dairy cattle, and deer. When the disease progresses to a certain extent, symptoms of enteritis accompanied by diarrhea are shown, and in addition, symptoms such as decreased weight gain, mastitis, decreased milk production, and decreased pregnancy rate may occur. Death may occur due to suppression of intestinal nutrient absorption, and since the incubation period is long and the pathogen can be excreted through feces without clinical symptoms, the disease occurs continuously in ranches once contaminated with Johne's bacteria, and therefore early screening and culling of the affected animals are important. In particular, clinical symptoms may appear in cattle aged 2 to 5 years depending on the individual's reaction to external factors such as primiparous acidity or severe stress and rearing in a poor environment. In sheep and goats, diarrhea is not obvious, but weight loss continues, and the disease progresses more acutely than in cattle, and there are many cases where there are no distinct lesions.

The pathogen of Johne's disease is Mycobacterium aviym subspices paratuberculosis , which latently infects the gastrointestinal tract wall and mesenteric lymph nodes in the body of living animals. Johne's bacteria discharged with feces are highly resistant to external environments such as temperature changes and dryness, and can survive in the natural environment for a long time in feces. Johne's bacteria are highly resistant to general disinfectants, but are sterilized within 10 minutes by formalin, carbolic acid, and cresol, and their sterilizing power is known to be reduced when mixed with organic matter such as feces.

Diagnosis of Johne's disease in cattle that have been euthanized or culled can be made by histopathological examination of the ileocecal region or regional lymph nodes, and culture and PCR of the organism from the ileum, lymph nodes, and feces. Diagnosis of Johne's disease in live cattle can be made by culture, PCR, intradermal reaction, complement binding reaction, and enzyme-linked immunosorbent assay, and a definitive diagnosis can be made at the clinical level in animals that exhibit typical clinical signs.

There are three main methods for diagnosing Johne's disease: enzyme-linked immunosorbent assay (ELISA) that measures antibodies, culturing bacteria from contaminated samples, and detecting bacteria genes in feces. Of these, ELISA is most commonly used to detect subclinical individuals because it is convenient and rapid, but it is insufficient to confirm the disease because of its low sensitivity and specificity. Therefore, the culture or gene detection method of Johne is accepted as the standard diagnostic method. However, the culture method is difficult to culture in the laboratory because the bacteria proliferate slowly in the body of infected animals due to the characteristics of the bacteria, so the probability of isolating and culturing the bacteria in actual subclinical cases is low at 45-72%, which requires a lot of time and money. The cPCR (conventional PCR) technique for detecting Johne's genes detects genes such as IS900, F57, ISMav02, and IS1311 that are unique to Johne. The cPCR technique requires repeated temperature changes for the three stages of denaturation, annealing, and extension for gene amplification. This requires expensive equipment such as a polymerase chain reactor (thermal cycler) and skilled personnel who can use it, making it difficult to apply in actual fields.

Loop-mediated isothermal amplification (LAMP) does not require temperature changes during the gene amplification process, so it only requires relatively inexpensive constant temperature equipment, and it is known to be more sensitive than the cPCR method because it uses six primers, unlike cPCR, which uses two primers. In addition, since color changes can be distinguished and diagnosed with the naked eye without separate equipment, the LAMP technique can be said to be suitable for field application.

As a prior art related to a method for detecting Johne's disease, Korean Patent Publication No. 10-2015-0117030 entitled "Composition and method for diagnosing Johne's disease comprising a Johne's disease-specific marker" discloses a composition for detecting whether a sample is infected with Johne's disease, the composition including a detection reagent for one or more biomarkers selected from the group consisting of ccl4, ccl5, cxcl12, fabp2, hp, pigr, abcb1, acp5, adm, c3, cd68, hgf, ikzf, il10, mapt, mmp9, nudt7, s100a12, serpine1, tg, and tnfsf13b, and a method for performing IS900 real-time PCR and ISMAP02 nested PCR to determine whether MAP is excreted from feces, and collecting blood from cattle and performing an ELISA test to examine individuals positive for blood antibodies.

In addition, Korean Patent Publication No. 10-2011-0042594, "MACS diagnostic primer pair and use thereof," discloses a MAC (Mycobacterium avium complex) diagnostic composition and a method for diagnosing MAC by performing PCR using a primer set specific to IS1311, IS901, IS900, and DT1 of MAC, and discloses a method for diagnosing MAC as Mycobacterium avium complex by performing multiplex PCR using the primer set, and then diagnosing it as Mycobacterium avium complex if a PCR product specific to IS900 is generated.

Since there are various subspecies with similar base sequences within the Mycobacterium species including Johne, it is important to perform the LAMP method using a base sequence specific to Johne. Meanwhile, LAMP primer sets for diagnosing Johne have been reported for the genes ISMap02 and IS900 (Sange et al., 2019; Trangoni et al., 2015), but a LAMP primer set for the IS1311 gene has not yet been reported.

Accordingly, the inventors of the present invention, while conducting research on a LAMP diagnostic method capable of detecting Bacillus cereus under optimal conditions, discovered a novel primer set sequence for IS1311 and a new primer set sequence for ISMap02, and established a composition and reaction conditions to which the same can be applied, and confirmed that the primer set exhibited significantly higher sensitivity and specificity than the existing standard diagnostic method, cPCR detection method, thereby confirming that the LAMP primer set of the present invention can be used to detect Bacillus cereus, thereby completing the present invention.

The purpose of the present invention is to provide a LAMP primer set for specifically detecting Johne bacteria and a method for detecting Johne bacteria using the same.

To achieve the above objectives,

The present invention provides a primer set for detecting Mycobacterium avium ssp paratuberculosis, comprising at least one primer set selected from the group consisting of a first primer set represented by the base sequence of SEQ ID NOs: 1 to 6; and a second primer set represented by the base sequence of SEQ ID NOs: 7 to 12.

In addition, the present invention provides a composition for detecting Yonephritis comprising the first primer set and the second primer set.

In addition, the present invention provides a kit for detecting Yone bacteria comprising the composition for detecting Yone bacteria.

In addition, the present invention comprises: 1) a step of isolating genomic DNA (gDNA) of a sample;

2) a step of amplifying the target sequence using the separated genomic DNA as a template and the primer set of clause 1; and

3) A method for detecting Mycobacterium avium ssp paratuberculosis is provided, comprising a step of detecting the amplified product.

The ISMap02 or IS1311 LAMP primer set for detecting Johne's disease of the present invention specifically detects only Johne's disease, exhibits improved sensitivity compared to existing diagnostic methods, and since the LAMP method does not require specialized personnel, it can be easily used in the field to diagnose Johne's disease.

Figure 1 is a diagram showing a partial sequence of the Yonebacterium gene.
Figure 2 shows the results of the specificity analysis for Y. melanogaster using the LAMP primer set (ISMap02).
Figure 3 shows the results of a specificity analysis for Y. melanogaster using a LAMP primer set (IS1311).
Figure 4 shows the results of sensitivity analysis for Y. melanogaster using the LAMP primer set (ISMap02).
Figure 5 shows the results of a sensitivity analysis for Y. melanogaster using a LAMP primer set (IS1311).

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set for detecting Mycobacterium avium ssp paratuberculosis, comprising at least one primer set selected from the group consisting of a first primer set represented by the base sequence of SEQ ID NOs: 1 to 6; and a second primer set represented by the base sequence of SEQ ID NOs: 7 to 12.

The term "Johne's Disease (Bovine paratuberculosis)" of the present invention is a chronic wasting digestive disease mainly occurring in ruminant animals such as cattle, sheep, goats, and deer, caused by Mycobacterium avium ssp paratuberculosis. Appetite is normal, but chronic diarrhea is shown, and death occurs due to malabsorption of nutrients and decreased weight gain caused by watery diarrhea. Since it is a bacterial infectious disease, the incubation period is long, 6 months to 2 years, and the causative agent is excreted through feces from stage 2 or higher of the disease, it is difficult to eradicate the disease once it occurs in a farm. Johne's disease is generally divided into an asymptomatic stage (stage 1-2) and a clinical stage (stage 3-4). Of these, stage 2 has no clinical symptoms, but since the bacteria are continuously transmitted to surrounding individuals, early detection and culling are important.

When infected with Johne's disease, it causes damage to the intestines of infected animals, resulting in persistent diarrhea, weight loss, and decreased conception rates, and eventually the infected animals die. This disease is distributed worldwide, and according to a 2007 USDA survey, it is estimated that 68% of dairy cows in the United States are infected. In Korea, the Johne's disease antibody positivity rate is reported to be between 6.1% and 16.4%, although there are some differences depending on the region. As such, Johne's disease is a problem even in developed countries, and economic losses cannot be avoided in dairy farms when infected. Furthermore, once Johne's disease occurs in a farm, it can take several years to eradicate it, so it is important to prevent the occurrence of Johne's disease by thoroughly managing the hygiene of the farm and calf management.

Animals infected with Johne's disease typically shed large amounts of the bacteria in their feces. Therefore, even if there is only one infected cow on a farm, Johne's disease can easily spread to young calves. Infected animals shed the bacteria in their milk and colostrum. Cows are most at risk of exposure to Johne's disease during the first few months of life. Calves can become infected from their mothers during pregnancy, but they can also become infected by ingesting contaminated colostrum, ingesting feces from infected cows on their teats, ingesting contaminated feed, or through contaminated environments or water. The pathogen that causes Johne's disease can survive in bedding, feces, and water for up to a year. Other animals, such as deer, sheep, and goats, can carry and shed the bacteria in their feces, so they can also spread Johne's disease when kept on farms with dairy cows.

The term " Mycobacterium avium ssp paratuberculosis " of the present invention refers to a pathogenic bacterium belonging to the genus Mycobacterium, known as the causative agent of Johne's disease that affects ruminants such as cattle. Johne's disease exists hidden in the mesenteric lymph nodes during the incubation period, and when the host's immunity is lowered by severe stress such as acetic acid, it proliferates and comes out through the intestinal wall and is excreted together with feces. The incubation period is long (2-3 years), and in the early stage of infection, there are no distinct clinical symptoms, and it is transmitted to other animals through the bacteria excreted in feces. Therefore, in order to effectively respond to the disease, it is essential to diagnose and isolate infected animals in the early stage of Johne's disease to prevent the spread of infection. In addition, because Johne bacteria have a slow growth rate and a specific cell wall structure containing mycoside C, mycolic acid, peptidoglycan, and lipopolysaccharide, they are highly resistant to chemical and physical treatments, making it very difficult to diagnose and control the disease.

Mycobacterium species include, in addition to M. avium subsp, paratuberculosis , M. avium subsp. avium ( MA ) , M. avium subsp. silvaticum ( MAS ), M. avium sub hominissuis ( MH ) , and M. intracellulare ( MI ) .

The above M. avium subsp. avium (MA) is also called avian tuberculosis or avian tuberculosis. The original host is birds, but it also infects humans, mainly infecting the lungs and creating multiple nodules, and the infection is not limited to the lungs. It spreads to the entire host (dessemination) and causes various diseases. It is a difficult bacterium to control, as it can withstand stomach acid without dying.

There has not been much research on the above M. avium subsp. silvaticum (MAS). In addition, it is known that almost all of them are similar to MAA and that they are isolated from nature only in extremely rare cases.

The above M. avium sub hominissuis (MH) is reported to be infected with more than half of M. avium patients. MH is also called pig type, and there is still no easy classification method for MA and MH. It is known to be less pathogenic than MA, but it is known to cause the most diseases in humans and is also difficult to treat.

The above M. intracellulare (MI) is a slow-growing bacterium that causes symptoms most similar to MA, and its characteristic is that it does not disseminate. It is similar to MA in all aspects, and the treatment is also the same as MA.

As described above, since various subspecies within the Mycobacterium species have similar base sequences, it is important to use a base sequence specific to Johne to detect Johne.

The primer set of the present invention is for isothermal amplification reaction (loop-mediated isothermal amplification reaction, LAMP).

The term loop-mediated isothermal amplification (LAMP) of the present invention refers to a method in which, unlike the existing PCR method, a PCR cycle is not required and a reaction is performed under isothermal conditions using a polymerase such as Bst or Gsp. Basically, four primers are used, both ends are synthesized into a loop structure to infinitely amplify a specific region of a gene, and the amplified DNA product is subjected to a precipitation reaction using a fluorescence detection reagent to confirm the presence or absence of amplification. Preferably, a fluorescence detection reagent can be used, but the invention is not limited thereto. Compared to the polymerase chain reaction (PCR) method, it has the characteristics of not requiring a denaturation reaction from one strand to two strands, the reaction proceeds at a temperature of 60 to 65°C, and no equipment such as a thermal cycler is required. In addition, the amplification speed is fast and the specificity is high, so it is possible to confirm whether the target has increased simply by observing the cloudiness of the reaction solution.

The primer set of the present invention is preferably used in an isothermal amplification reaction (LAMP; Loop-mediated isothermal amplification), wherein the first primer set targets ISMap02 of Johne, the second primer set targets IS1311 of Johne, and each primer set is composed of an internal primer set, an external primer set, and a loop primer set. Specific information of each primer is as follows.

F3 of sequence number 1 constituting the first primer set is a forward outer primer of the ISMap02 gene;

B3 of sequence number 2 is the backward outer primer of the ISMap02 gene;

FIP of sequence number 3 is the forward inner primer of the ISMap02 gene;

BIP of sequence number 4 is the backward inner primer of the ISMap02 gene;

LF in sequence number 5 is the loop forward primer of the ISMap02 gene;

LB in sequence number 6 is a loop backward primer of the ISMap02 gene.

F3 of sequence number 7, which constitutes the second primer set, is a forward outer primer of the IS1311 gene;

B3 of sequence number 8 is the backward outer primer of the IS1311 gene;

FIP of sequence number 9 is the forward inner primer of the IS1311 gene;

BIP of sequence number 10 is the backward inner primer of the IS1311 gene;

LF in sequence number 11 is the loop forward primer of the IS1311 gene;

LB of sequence number 12 is a loop backward primer of the IS1311 gene.

The term "primer" of the present invention refers to a single-stranded oligonucleotide which can act as a starting point for template-directed DNA synthesis under appropriate conditions (e.g., four different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer solution and at an appropriate temperature.

The appropriate length of the above primer may vary depending on the intended use, but is typically 15 to 30 nucleotides. Shorter primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence does not need to be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.

The primer of the present invention can be chemically synthesized using a method known in the art, such as, for example, a phosphoramidite solid support method. In addition, it can be modified by methylation or the like using a known method. In addition, the primer of the present invention can, if necessary, include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g., 32P), fluorescent molecules, chemical groups (e.g., biotin), and the like.

The term “outer primer” of the present invention refers to a single-stranded oligonucleotide that binds to template DNA at a position further outside the position where the inner primer binds to the template DNA, and after the inner primer binds to the template DNA and the DNA chain is extended, strand displacement occurs due to the binding of the outer primer to the template DNA, causing the previously formed chain to fall off. The primers of sequence numbers 1 and 2 of the present invention are outer primers located inside the ISMap02 gene to be amplified, wherein the primer of sequence number 1 is a forward outer primer (F3) and the primer of sequence number 2 is a backward outer primer (B3). In addition, the primers of sequence numbers 7 and 8 are outer primers located inside the IS1311 gene to be amplified, wherein the primer of sequence number 7 is a forward outer primer (F3) and the primer of sequence number 8 is a reverse outer primer (B3).

The term “inner primer” of the present invention refers to a single-stranded oligonucleotide that can bind to template DNA and act as a starting point for the synthesis of a new DNA chain. The primers of SEQ ID NOS: 3 and 4 of the present invention are inner primers located within the ISMap02 gene to be amplified, wherein the primer of SEQ ID NO: 3 is a forward inner primer (FIP) and the primer of SEQ ID NO: 4 is a backward inner primer (BIP). In addition, the primers of SEQ ID NOS: 9 and 10 are inner primers located within the IS1311 gene to be amplified, wherein the primer of SEQ ID NO: 9 is a forward inner primer (FIP) and the primer of SEQ ID NO: 10 is a reverse inner primer (BIP).

The term “loop primer” of the present invention refers to a single-stranded oligonucleotide that can act as a starting point for nucleotide synthesis, which allows the initial stem loop structure chain formed when the inner and outer primers bind to template DNA to increase the number of times the loop structure is formed, thereby accelerating the overall reaction. The primers of SEQ ID NOS: 5 and 6 of the present invention are loop primers of the ISMap02 gene to be amplified, wherein the primer of SEQ ID NO: 5 is a forward loop primer (LF) and the primer of SEQ ID NO: 6 is a backward loop primer (LB). In addition, the primers of SEQ ID NOS: 11 and 12 are loop primers of the IS1311 gene to be amplified, wherein the primer of SEQ ID NO: 11 is a forward loop primer (LF) and the primer of SEQ ID NO: 12 is a reverse loop primer (LB).

The ISMap02 and IS1311 genes of the present invention are genes specific to Mycobacterium avium ssp paratuberculosis (MAP).

According to one embodiment of the present invention, when an isothermal amplification reaction is performed using a primer set according to the present invention, compared to existing detection methods, Streptococcus pneumoniae can be specifically detected in a sample with high sensitivity, the results can be quantified, and the reaction time is greatly improved, so that the level of contamination and occurrence by Streptococcus pneumoniae can be quickly and accurately confirmed, and since no specialized technology is required, it can be effectively utilized as an early screening system for detecting Streptococcus pneumoniae in livestock farming fields.

The present invention provides a composition for detecting Yonephritis, comprising the first primer set and the second primer set.

The composition for determination of the present invention may comprise one or more other component compositions, solutions or devices suitable for the analytical method.

The present invention provides a kit for detecting Yone bacteria comprising the composition for detecting Yone bacteria.

The kit of the present invention may include typical components included in a microbial/gene detection kit together with the primer set for detecting the above-described Johne bacteria. The typical components included in the kit for detecting the above-described Johne bacteria may include, but are not limited to, a reaction buffer, DNA polymerase, dNTP, and MgCl2.

Meanwhile, the components included in the kit may be manufactured in a liquid form, or may be manufactured in a dried form to improve the stability of the product by reducing the degree of freedom of the included components. For such manufacturing in a dried form, a drying step must be applied, and at this time, heating drying, natural drying, reduced pressure drying, freeze drying, or a combination of these processes may be used.

The present invention comprises the steps of: 1) isolating genomic DNA (gDNA) of a sample;

2) a step of amplifying the target sequence using the separated genomic DNA as a template and the primer set of clause 1; and

3) A method for detecting Mycobacterium avium ssp paratuberculosis is provided, comprising a step of detecting the amplified product.

In the above detection method, the sample of step 1) means an unknown material from which DNA required to confirm the presence of the Johne gene is extracted, and includes, but is not limited to, food contaminated with Johne, natural products (e.g., water), biological samples, etc.

The above biological samples include, but are not limited to, blood, sweat, urine, feces, tissues, etc. isolated from an individual.

The above entities include all species that can be infected with the bacterium Johne, including but not limited to dogs, cattle, pigs, sheep, whales, chickens, and ducks.

In the above detection method, the sample of step 1) may be a mixed sample, and the mixed sample may be a sample capable of detecting and identifying the bacterium Johne before the separation of the bacterium is completed, preferably by targeting the ISMap02 or IS1311 gene of the bacterium Johne, and may be an enriched solution of a sample mixed with a mixture, such as feces or an environmental sample, rather than a pure culture solution in which the bacterium has been separated.

In the above step 2), the target sequence can be amplified by performing an isothermal amplification reaction (loop-mediated isothermal amplification reaction, LAMP).

The isothermal amplification reaction of step 2) above can be performed at 60 to 70°C for 1 to 2 hours, and preferably can be performed at 65°C for 1 hour.

In the above detection method, detection of the amplification product of step 3) can be performed through, but is not limited to, a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement.

In the above step 3), the presence or absence of the detection of the Yone bacteria can be determined using the change in color of the amplified product.

In the above step 3), if the color of the amplified product is blue, it can be judged as positive for Johne bacteria.

In the above step 3), if the color of the amplified product is purple, it can be judged as negative for Johne bacteria.

The above detection method may further include a step of quantifying the bacterium by measuring the detection time of step 3).

The above “quantification” may be quantifying the detection time of a fluorescent substance according to the DNA concentration by comparing it with a standard curve when performing an amplification reaction using a primer set according to the present invention, and preferably, the DNA concentration may be quantified by measuring the detection time of a fluorescent substance by detecting the amplification product of step 3) using a fluorescent indicator.

In a specific embodiment, the inventors collected strains by isolating the bacteria from the ileum tissue of a Johne-positive individual, and extracted genomic DNA from purely cultured Johne strains. To verify the specificity of the LAMP primer set, one standard strain of Johne (K-10), one field strain (SU-29), and one commensal strain (C. Pseudotuberculosis), as well as three Mycobacterium species (MA, MI, MH) and three commensal strains (Escherichia coli, Staphylococcus aureus) were cultured.

In addition, in order to design a set of specific primers (ISMap02, IS1311) for the detection of Johne through LAMP reaction, the gene sequence specific to Johne was identified through in-silico analysis (see Fig. 1), and external primers (F3, B3), internal primers (FIP, BIP), and loop primers (LF, LB) that recognize six target sequences among the sequences were created using the NEB LAMP primer design program.

In addition, an isothermal amplification reaction was performed using the above primers, and the presence or absence of the Yone bacteria was observed through a color change with the naked eye. If positive (+), it was confirmed as blue, and if negative (-), it was confirmed as purple (see Figures 2 to 5).

In addition, in order to verify the specificity of the primer set for the diagnosis of Johne bacteria LAMP, the specificity was analyzed for a total of 11 strains including 1 standard strain of Johne bacteria, 4 strains of Johne bacteria isolated in the field, 3 strains that are different subspecies of the same species as Johne bacteria, and 3 strains of other commensal bacteria existing in the environment. By confirming that both the ISMap02 and IS1311 primer sets showed positive reactions only in the MAP standard strain and the field isolated strains, it was confirmed that the primer set of the present invention can specifically detect Johne bacteria (see Figs. 2 and 3).

In addition, as a result of verifying the sensitivity of the above primer sets, it was confirmed that both LAMP primer sets (ISMap02, IS1311) of the present invention can detect Johne bacteria up to 0.1 pg/㎕, and that the sensitivity is 10 times higher than that of the conventional PCR (IS1311, ISMav02), which is a standard diagnostic method (see Figures 4 and 5).

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the claims also fall within the scope of the present invention.

<Example 1> Design of a set of specific primers (ISMap02, IS1311) for diagnosis of Y. y. LAMP

In order to detect Bacillus subtilis, the causative agent of Johne's disease, through the LAMP reaction, the gene sequence specific to Bacillus subtilis was identified through in-silico analysis. Specifically, candidate genes for MAP diagnosis were selected through a review of existing literature, and the sequences of the corresponding genes were downloaded from the NCBI database. Using ClustalW and Mega7 software, the candidate genes for MAP diagnosis (IS1311, ISMAV02) were compared with the gene sequences of MAP and closely related Mycobacterium species, and the gene sequence region with high specificity for MAP was identified. After that, the sequence was entered into Primer explore 5 and NEB LAMP primer design software, and external primers (F3, B3), internal primers (FIP, BIP), and loop primers (LF, LB) that recognize six target sequences were created. A schematic diagram and specific sequence of each primer target region in the IS1311 and ISMAV02 sequences are shown in Figure 1, and the complementary LAMP primer sequences are shown in Table 1.

gene designation Primer sequence (5'-3') ISMap02 F3 AGCGTGAGTTGTTGGATGC B3 CCACGGTCTCGTTATCCGA FIP GAAACAACTGGCTGCGGTGCGTCGGTTGCCGGGCATC BIP CGGACTTGTTCCCGTCGCAGCGCAGGGCCTGCAAGGT LF ACATGCTGTCGGCCTTCAGA LB AGGTGATGGCCTCGGTGAT IS1311 F3 AGACCACCCGATGGTTGA B3 TGCACGGCACCTCCAC FIP CGGCCTCTATCCGAACAGCACTCCCGAGCCCTTGCAGTA BIP CGGTGTCCAGATCGGCGCAAAACGATTTGGTCAAAGCCCTC LF CCTATGTGTTCCTCGACGCC LB CCGGGAGACCTCGCTTTTG

<Experimental Example 1> Verification of the specificity of the primer set for the diagnosis of Johne's bacterium LAMP

Since there are various subspecies with similar base sequences within the Mycobacterium species including Johne, it is important to perform the LAMP method using a base sequence specific to Johne. Therefore, in this study, a total of 11 strains, including one standard strain of Johne, four strains of Johne isolated from the field, three strains that are different subspecies of the same species as Johne, and three strains of commensal bacteria existing in other environments, were collected and analyzed for specificity.

<1-1> Extraction of genome DNA from suspected Johne bacteria infection samples

To verify the specificity and sensitivity of the LAMP primer set of Example 1, the field strain of Yone was collected by isolating the strain from the ileum tissue of a Yone-positive individual using 7H9 medium. The purely cultured Yone colony was transferred to a 1.5 ml tube, 180 ㎕ of ATL buffer (tissue lysis) and 20 ㎕ of Proteinase K were added, and the mixture was mixed until sufficiently disrupted (voltexing), and then the mixture was incubated at 56°C for at least 2 hours to perform the lysis process. After that, 200 ㎕ of AL buffer (lysis) was added and mixed, and the mixture was incubated at 70°C for 10 minutes, after which 200 ㎕ of ethanol was added and mixed until dissolved. The mixture was placed into a Dneasy Mini spin column and centrifuged (8,000 rpm, 1 min). The lower tube was discarded and transferred to a new tube. 500 ㎕ of AW1 buffer (wash) was added and centrifuged (8,000 rpm, 1 min). The lower tube was discarded again and transferred to the same tube. 500 ㎕ of AW2 buffer (wash) was added and centrifuged (14,000 rpm, 3 min). To remove moisture from the Dneasy Mini spin column membrane, the filtrate in the lower tube was emptied and centrifuged once more in the same tube (14,000 rpm, 1 min). Finally, the Dneasy Mini spin column was transferred to a 1.5 ㎖ tube, 50 ㎕ of AE buffer (elution) was added, and the mixture was left at RT for about 2 minutes and centrifuged (8,000 rpm, 1 min) to extract genomic DNA.

<1-2> Strain distribution and genome DNA extraction

To verify the specificity and sensitivity of the LAMP primer set of Example 1, one standard strain of C. pyogenes (K-10), one field strain (SU-29), and one commensal strain (C. pseudotuberculosis) were donated by Seoul National University and Chonbuk National University College of Veterinary Medicine, respectively, and three Mycobacterium species (MA, MI, MH) and three commensal strains (Escherichia coli, Staphylococcus aureus) were provided through the Korea Veterinary Biobank (KVCC), cultured, and genomic DNA was extracted using the same method as described above.

Information about the above strains is given in Table 2 below.

Strain Strain name etc Mycobacterium avium subsp. paratuberculosis (MAP) K-12 Representative variant Mycobacterium avium subsp. paratuberculosis (MAP) SU-29 separation Mycobacterium avium subsp. paratuberculosis (MAP) C15-25 separation Mycobacterium avium subsp. paratuberculosis (MAP) 20-13 separation Mycobacterium avium subsp. paratuberculosis (MAP) T19-13 separation Mycobacterium avium subsp. avium(MA) W-986LN Representative variant Mycobacterium avium subsp. Intracellulare (MI) D-100 Representative variant Mycobacterium avium subsp. hominissuis(MH) C-29 Representative variant C.Pseudotuberculosis JBNU cp-001 Representative variant Escherichia coli O157:H7 Representative variant Staphylococcus aureus 23235 Representative variant

<1-3> LAMP reaction conditions

For LAMP reaction, 0.2 μM F3 and B3 primers, 1.6 μM FIP and BIP primers, 0.4 μM LF and LB primers, Isothermal Amplification Buffer II (2.5 μl), Bst2.0 WarmStart DNA Polymerase (1 μl. 8 U) (New England Biolabs), 1.4 mM dNTP (Thermo), 6 mM MgSO4, 0.8 M betaine (Sigma), and 10 ng template DNA (1 μl) were mixed to a total volume of 25 μl, and the amplification reaction was performed at 65°C for 1 hour and then terminated at 80°C for 5 minutes.

The presence or absence of Yone bacteria was observed through color change with the naked eye. Positive (+) was observed as blue, and negative (-) was observed as purple (Figs. 2 to 5).

<1-4> Validation of the specificity of the primer set for the diagnosis of Johne's disease LAMP

Using the LAMP primer set of Example 1 above, LAMP reaction was performed on 11 strains. As a result, MAP standard strain and field isolated strain showed positive reaction (purple), and the remaining Mycobacterium subspecies and commensal bacteria showed negative reaction (blue). All products were electrophoresed on a 1.5% agarose gel containing Redsafe for 30 minutes, and a specific band was confirmed under UV, etc.

Through the above experiments, it was confirmed that the LAMP primer set for ISMap02 and IS1311 of the present invention can specifically detect only Y. pyogenes (Fig. 2).

<Experimental Example 2> Sensitivity Verification of the Primer Set for Yonebacterium LAMP Diagnosis

In order to verify the sensitivity of the LAMP primer set of Example 1, the genomic DNA concentration of the standard strain sample of Johne was diluted to 1 ng/㎕, 0.1 ng/㎕, 10 pg/㎕, 1 pg/㎕, 0.1 pg/㎕, 10 fg/㎕, or 1 fg/㎕, and the LAMP reaction was performed in the above-mentioned manner. As a result, it was confirmed that both LAMP primer sets of the present invention (ISMap02, IS1311) could detect Johne up to 0.1 pg/㎕ (Figs. 4 and 5). This is a result that is 10 times higher in sensitivity than IS1311 and 100 times higher than ISMav02, compared to the conventional PCR (IS1311, ISMav02), which is a standard diagnostic method recommended by OIE.

As described above, it was confirmed that the ISMap02 and IS1311 LAMP primer sets of the present invention specifically detect Johne and have a sensitivity that is 10 times higher than that of the standard diagnostic method, cPCR, thereby confirming that the primer sets of the present invention can be used to detect Johne.

Attach electronic file of sequence list

Claims (15)

A first primer set represented by the base sequence of sequence numbers 1 to 6; and
A primer set for detecting Mycobacterium avium ssp paratuberculosis , comprising at least one primer set selected from the group consisting of a second primer set represented by a base sequence of sequence numbers 7 to 12. In the first paragraph,
A primer set for detecting Yonephritis, characterized in that the above primer set is for isothermal amplification reaction (loop-mediated isothermal amplification reaction, LAMP). In the first paragraph,
A primer set for detecting Johne, characterized in that the first primer set is specific for ISMap02 of Johne. In the first paragraph,
The second primer set is a primer set for detecting Johne, characterized in that it is specific for IS1311 of Johne. A composition for detecting Mycobacterium avium ssp paratuberculosis, comprising the primer set of claim 1. A kit for detecting Mycobacterium avium ssp paratuberculosis, comprising the composition of claim 5. 1) A step of isolating genomic DNA (gDNA) of the sample;
2) a step of amplifying the target sequence using the separated genomic DNA as a template and the primer set of clause 1; and
3) A method for detecting Mycobacterium avium ssp paratuberculosis , comprising a step of detecting the amplified product. In Article 7,
A method for detecting Yonephritis, characterized in that in the above step 1), the sample is at least one selected from the group consisting of blood, sweat, urine, feces, and tissue separated from an individual. In Article 7,
A method for detecting Yone bacteria, characterized in that in the above step 2), the target sequence is a target sequence for the ISMap02 or IS1311 gene. In Article 7,
A method for detecting Yone, characterized in that in step 2), a target sequence is amplified by performing an isothermal amplification reaction (loop-mediated isothermal amplification reaction, LAMP). In Article 10,
A method for detecting Yone bacteria, characterized in that the above isothermal amplification reaction is performed at 60 to 70°C. In Article 11,
A method for detecting Yone bacteria, characterized in that the above isothermal amplification reaction is performed for 1 to 2 hours. In Article 7,
A method for detecting Streptococcus pneumoniae, characterized in that in step 3), the presence or absence of Streptococcus pneumoniae is determined by using a change in the color of the amplified product. In Article 13,
A method for detecting Johne bacteria, characterized in that if the color of the amplified product is blue, it is judged to be positive for Johne bacteria. In Article 13,
A method for detecting Johne bacteria, characterized in that if the color of the amplified product is purple, it is judged to be negative for Johne bacteria.