KR20110028689A - Detection method of orientina thstsugamushi using high orientina thstsugamushi specific primers with specific structure of a reverse complementary sequence - Google Patents

Abstract

PURPOSE: A primer for detecting tsutsugamushi disease infection and a detection method are provided to quickly and easily determined the infection. CONSTITUTION: A primer for detecting Orientia tsutsugamushi infection comprises bases of sequence numbers 1-4. A kit for detecting Orientia tsutsugamushi infection contains the primer of sequence numbers 1-4. A method for detecting Orientia tsutsugamushi comprises: a step of isolating DNA from a test sample; a step of performing nested-PCR using the DNA and the primer of sequence numbers 1-4; and a step of analyzing the PCR product.

Description

Detection method of Orientina thstsugamushi using high Orientina thstsugamushi specific primers with specific structure of a reverse complementary sequence}

The present invention relates to a method for detecting an infection of Orientia tsutsugamushi , a disease causing agent that causes tsutsugamushi disease in humans using a polymerase chain reaction (PCR). Specifically, primers having a characteristic structure capable of detecting the Orientia Tsutsugamu while providing improved PCR amplification specificity, and infection of the Orientia Tsutsugamu through PCR using these primers The present invention relates to a PCR detection method capable of detecting whether or not the present invention relates to a detection technique that can be utilized for diagnosing infection of Orientia Tsutsugamu bacteria.

Tsutsugamushi disease, also called scrub typhus, scrub typhus or caterpillar disease, is known as a sclero typhus, which was found in the hair mite larva when the mediator, Trombiculidae chigger, adheres to human skin and aspirates fluids. Orientia tsutsugamushi is infected as it enters the body. Symptoms include cold chills, fever and headache above 40 ° C, sudden cough, vomiting, sore throat, and sore throat. Spots and papular rashes appear on the chest, abdomen, torso or lower extremity and rarely on the face, palms or soles on days 3-7 of the onset, which last 2 to 10 days. Hair mite bites produce eschar covered with black scab within days of onset, and in most cases there are no symptoms such as pain or itching. Crust is observed in 70-90% of Tsutsuga's patients. Some patients have no skin, short fever, and more skin rashes. In some cases, symptoms such as cranial bands, conjunctivitis, and severe cases of consciousness, pneumonia, and circulatory disorders may occur. The severity of these clinical symptoms varies from person to person and depends on the difference in serotypes of causative organisms prevalent in the region. Tsutsugamushi has long been known as an endemic disease in Japan, and occurs in a wide range of regions including Korea, China, Thailand, India, Pakistan, Malaysia, the Philippines and Australia. In Korea, there was a report of the first case of the UN armed forces in 1951, and about 200 cases occurred each year from 1994 to 1997, then increased to 1,140 in 1998, and increased to 2,638 in 2001 and 4,699 in 2004. 6057 people are on a steady increase. It usually occurs in the period from mid-October to early December.

Orientia Tsutsugamu, the causative agent of Tsutsugamosis, has three main strains, Gilliam, Karp, and Kato, depending on the antigen. Recently, new strains in Japan, Thailand, and Korea Some have been reported. Orientia Tsutsugamushi is a monococci with a diameter of 0.5-0.7 μm and a length of 1.2-2.5 μm, and can be easily observed by optical microscopy after laver staining. Orientia Tsutsugamushi's constituent polypeptides (polypeptides) were analyzed by SDS-PAGE, about 30 polypeptide bands appeared. The major polypeptides are 110, 80, 70, 60, 56, 46, 42, 35, 28, 25 kDa. Of these, the 56 kDa polypeptide is a type-specific antigen (TSA) and is the most abundant major constituent polypeptide among the polypeptides constituting Orientia Tsutsugashishi. The 56 kDa polypeptide has a type-specific antigenicity as a major immunoantigen, and it can be seen that it is present in the outermost layer because it is easily degraded by trypsin treatment. Orientia Tsutsugamushi is not cultured in artificial media. Cell culture and fertilized egg culture are mainly used. Orientia Tsutsugamu can also be subcultured by inoculating susceptible animals such as mice.

Indirect immunofluorescence and immunoassay methods are the most widely used diagnostic methods for Tsutsuga's disease.These two methods have the advantage of distinguishing early infection from reinfection with high sensitivity and specificity. It is the recommended method. On the other hand, indirect immunofluorescence antibody test requires a facility for tissue culture or egg hatching in order to obtain the necessary antigen, and has the disadvantage that can be tested only in a laboratory equipped with a fluorescence microscope. In addition, immunoassay methods often have the disadvantage of being false-positive. In addition, all of the methods used for such serological diagnosis require a large amount of antigens, and there is a disadvantage in that culturing of bacteria to obtain necessary antigens is necessary. These conventional inspection methods have a problem that the inspection process is complicated and time consuming, and requires precision instruments to process the sample and read the results, and also requires highly skilled expertise. Therefore, there is a need for a new detection method that is faster and easier to determine.

PCR-based detection methods have been proposed as an easy alternative diagnosis method. PCR is a molecular biological method that can exponentially amplify the number of copies of a desired portion of DNA. Any portion of the DNA can be amplified by PCR only by knowing its sequence. PCR was first invented by K. Mullis in the mid-1980s and has been used in many biology-related studies, including molecular genetics, to study and analyze genes, and is still widely used today. Recently developed PCR techniques include rapid PCR, which reduces the time required for amplification, direct PCR, reverse transcription, and PCR, which eliminates the purification of samples. Reverse transcriptase PCR (RTR-PCR), which combines RNA to be used as a template, real-time PCR, primary PCR and 2 for real-time monitoring of PCR reactions Two-step PCR consists of nested-PCR, which improves sensitivity, and multiplex PCR, which simultaneously amplifies multiple target genes. In addition, a number of PCR techniques and PCR-related techniques have been developed. Detailed description thereof will be omitted. PCR is currently used in a variety of research and application areas, and has become a powerful experimental technique for most biological research fields (Science 252: 1643-1651, 1991).

PCR-based detection techniques for bacteria or viruses that determine whether bacteria or viruses with their genomes are present in the sample through detection of the presence of a genome in the sample It has become a common technology and is very widely used now. Such PCR-based detection of bacteria or viruses is actively used in the field of disease diagnosis for the purpose of diagnosing infectious diseases related to bacteria or viruses (Virol. Methods 66: 39-50, 1997; Clin.Microbiol 35: 2076-2082, 1997). PCR diagnosis, which is a diagnostic method based on the genome of an infectious agent, can provide a very high diagnostic sensitivity compared to other diagnostic methods due to the inherent characteristic of PCR that can generate a large number of amplification products from the PCR template genome. Due to the fact that it can provide high sensitivity and the time required for diagnosis is very short compared to other methods, PCR-based diagnostic methods are gradually replacing other diagnostic methods.

As a PCR-based prior art related to Tsutsuga's disease, the following inventions can be suggested. Korean Patent Registration No. 10-0657866 discloses a method for detecting erythema rickettsia bacteria using Nested-PCR. In the above invention, the gene of Rickettsia's outer membrane protein B (romp B) is targeted for detection. In addition to the detectable bacteria according to the invention described has rikechiah fertilization Rica (Rickettsia sibirica), rikechiah Akari (Rickettsia akari), rikechiah nose nori (Rickettsia conorii), rikechiah Felice (Rickettsia felis) and rikechiah japonica (Rickettsia japonica). However, the present invention is not suitable for the detection of Orientia Tsutsugamu bacteria. Korean Patent Publication No. 10-2008-0111353 discloses a microbial detection method using PCR and single strand conformation polymorphism (SSCP) (hereinafter referred to as PCR-SSCP method). In the present invention, 16S ribosomal RNA (rRNA), translation elongation factor 2 (EF-2), translation initiation factor 3 (IF-3), and translation initiation factor 2 (IF-2) are used as a target gene of PCR. Korean Patent Registration No. 10-0550463 discloses a primer for the simultaneous detection of rickettsia and Tsutsugamu bacteria. In the above invention, the groEL gene encoding the GroEL protein is used as a target gene for PCR. For reference, the invention similar to Korean Patent Registration No. 10-0550463 in that it targets the groEL gene encoding GroEL protein is Korean Patent Registration No. 10-0681837, Korean Patent Registration No. 10-0553520, and Korean Patent Patent Registration No. 10-0544069, Republic of Korea Patent Registration No. 10-0550463 and Republic of Korea Patent Registration No. 10-0681820.

The inventors of the present invention have tried to develop a method capable of detecting an infection with Orientia Tsutsugamu, which is simple, inexpensive, and sensitive and specifically compared to the conventional methods described above. Primers with specific structures that are specific for the type-specific antigen (TSA) gene and can significantly reduce non-specific amplification in its amplification, and nested-PCR-based Orientia Tsutsugamu infection The present invention has been completed by developing a detection method. The 56 kDa type specific antigen is mainly used immunologically. Examples include US Pat. No. 6,482,415, US Pat. No. 6,699,674, and US Pat. No. 7,335,477, which do not disclose detection of Orientia Tsutsugamu based on nested-PCR. PCR detection of genes encoding a 56 kDa type specific antigen of Orientia Tsutsugamu has been published in several papers (J. Med. Entomol. 38: 308-311, 2001; Clin. Med. J. 115: 1881-1882, 2002; Am. J. Trop. Med. Hyg. 54: 647-651, 1996). However, these prior arts have a problem of low specificity and sensitivity in the PCR step. We solved this by using primers that could improve specificity.

Throughout this specification, numerous papers and patent documents are referenced and their citations are indicated in parentheses. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

Therefore, an object of the present invention is to solve the problems of the prior art and the technical problem that has been requested from the past.

An object of the present invention is to overcome the limitations of specificity and sensitivity that the existing technology can provide, primers that can be used to test the infection of Orientia Tsutsugamu with improved specificity and sensitivity, a test kit comprising the same and using the primers It is to provide a method for detecting the presence of Orientia Tsutsugamu City.

In order to solve the problems of the present invention as described above,

The present invention is a primer capable of amplifying a partial sequence of a gene encoding a 56 kDa type specific antigen polypeptide of Orientia tsutsugamushi , which is a part from the 5'-end of the 5'-end of the primer. Provided is a primer for detecting an Orientia Tsutsugamu infection, characterized in that the complementary sequence of the reverse of the base sequence up to.

The primer is preferably composed of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 4.

In another aspect, the present invention provides a kit for detecting the infection of Orientia Tsutsugamu comprising the primer.

In another aspect, the present invention provides a kit for diagnosing Orientia Tsutsugamushi-related disease, comprising the primer.

In another aspect, the present invention provides a method for testing the infection of Orientia Tsutsugamu using the primer.

The inspection method,

(1) extracting DNA from a sample;

(2) performing nested-PCR using a primer combination consisting of SEQ ID NO: 1 to SEQ ID NO: 4 and the extracted DNA;

(3) It is preferable to include the step of investigating the infection of Orientia Tsutsugamu through the analysis of the PCR product obtained from step (2).

The present invention allows for quick and easy determination of infection with Orientia Tsutsugamu with improved specificity and sensitivity compared to conventional methods. Therefore, the present invention can be usefully used as a detection method for Orientia Tsutsugamu, and can be effectively used for development of products for diagnosing Orientia Tsutsugamushi-related diseases.

As described above, the present invention provides specific primers capable of binding to the genome of Orientia Tsutsugamu strain, and provides a kit for detecting the presence of Orientia Tsutsugamu infection comprising the same, and a kit for diagnosing Orientia Tsutsugamushi related diseases, Furthermore, the present invention provides a method for testing the infection of Orientia Tsutsugamu based on Nested-PCR using these primers.

Specifically, the present invention is a specific primers that can be used for primary PCR amplification from a gene encoding a 56 kDa size specific antigen of Orientia Tsutsugamu. Provided primer sequences consisting of the nucleotide sequence shown in 2. In addition, the present invention is a specific primers that can be used to specifically amplify the second nucleotide sequence located in the interior of the first PCR product of the base sequence preferably in the nucleotide sequence shown in SEQ ID NO: 3 to SEQ ID NO: 4 Provided primer sequences were made. These specific primers have a reverse complementary sequence introduced at the 5′-terminus of each primer to reduce non-specific amplification and improve sensitivity. These specific primers are a portion from the 5'-end site of the existing primer sequence, preferably the 3-7th base from the 5'-start, more preferably SEQ ID NO: 1 To a complementary sequence complementary to the fifth base from the 5′-start, such as the nucleotide sequence set forth in SEQ ID NO: 4. This reverse complementarity sequence allows primers to remain inactive for a period of time prior to entering the full-scale reaction of PCR, including preparing a PCR reaction solution. Inactivation of the primer is realized by the primers being bound to each other during this period, thereby inhibiting their role as primers (binding to the template to be the starting point of amplification). That is, the primers of the present invention can be bound to the template only at the annealing temperature in the normal PCR reaction after the template denaturation step, thereby increasing specificity in PCR amplification. In general, specific binding of the template and the primer is ensured at the annealing temperature. When using ordinary primers (primary primers without reverse complementary sequences), the binding between the template and the primers is nonspecific below the annealing temperature, which results in nonspecific amplification. In addition, the introduction of the complementary sequence of the inverse is amplification dependent on the PCR product generated before the third cycle from the third cycle in the PCR reaction cycle, compared to the amplification depending on the first template (the initial template). This prevails. As a result, the specificity is also improved by using a PCR product composed of a much simpler sequence as a template, as compared to when the template is composed of the original template including several parts of the sequence which are not necessary. On the other hand, the number of sites for primer priming to the template is increased by the number of complementary sequences of the reverse introduced, resulting in improved sensitivity. Specifically, the primers set forth in SEQ ID NO: 1 to SEQ ID NO: 4 of the present invention are the first primer sequence consisting of a priming sequence to bind the PCR template and a portion from the 5'-end of the 5'-end of the original primer sequence. Complementary sequences complementary to are the modified primers additionally introduced at the 5'-end of the original primer.

Hereinafter, the content of the present invention will be described in more detail, but it will be understood by those skilled in the art that the present invention may be variously modified and changed without departing from the spirit and scope of the present invention.

Specific primers in the present invention target amplification of a gene region encoding a 56 kDa type specific antigen (TSA) polypeptide of Orientia Tsutsugamu. A diagram showing the sites to be amplified in each PCR is shown in FIG. 1.

More specifically, the specific primers of the present invention are classified into primary PCR amplification primers and secondary PCR amplification primers, and primary PCR amplification primers generate primary PCR products through PCR from DNA, and secondary PCR. Amplification primers are used to amplify specific gene sites through secondary PCR based on the primary PCR product.

Specific primers capable of detecting the Orientia Tsutsugamu can be implemented through a primer combination of SEQ ID NO: 1 to SEQ ID NO: 4. The specific primer combination consists of one primary PCR forward primer, one primary PCR reverse primer, and one secondary PCR forward primer and one secondary PCR reverse primer, and the size of the primary PCR amplification product. Is 1,156 bp to 1,168 bp and the size of the second PCR amplification product is 448 bp to 471 bp.

The base sequences of the respective primers in the specific primer combinations used in the primary PCR and the secondary PCR of the present invention are as follows. The forward primer for primary PCR amplification is 5'- GCA AT A TTG CTA GTG CAA TGT CTG C-3 '(25-mer, SEQ ID NO: 1), and the reverse primer for primary PCR amplification is 5'- ATG CA T GCA TGR CGC TKC AAT TTA-3 '(24-mer, SEQ ID NO: 2). The forward primer for secondary PCR amplification was 5'- ATA GG C CTA TAA GTA TWG CKG ATC G-3 '(25-mer, SEQ ID NO: 3) and the reverse primer for secondary PCR amplification was 5'- CAT CT A GAY. GCA CTA TTA GGC AAA-3 ′ (24-mer, SEQ ID NO: 4). These primers are modified primers in which a sequence complementary to a portion from the 5'-end of the 5'-end of the original primer sequence is further introduced at the 5'-end of the original primer. Additional complementary sequences of the introduced stations are underlined. In this specification, IUB ambiguity codes are used to indicate the sequence of primers. Therefore, in the primer sequence, A represents adenine, G represents guanine, C represents cytosine, T represents thymine, W represents a possible site of both adenine and thymine, and K Represents a possible site for both guanine and thymine, R represents a possible site for both adenine and guanine, and Y represents a possible site for both cytosine and thymine. The same applies to the presentation of all sequences herein.

Each of the specific primers of the present invention may be altered a part of the constituent base, but the characteristics of having a reverse complementary sequence should be maintained. Such alterations can be implemented as partial deletions, additions, substitutions, or combinations of constituent base sequences, but such alterations in nucleotide sequences can result in significant disruption in specific binding to the template of the primer. Requires attention. Therefore, it should be determined empirically or empirically within the scope of not impairing specificity. Since this is a general matter to those skilled in the art, a detailed description thereof will be omitted. Most preferred is to maintain the sequence of primers set forth in the present invention.

The orientia Tsutsugamu detection method using the specific primers of SEQ ID NO: 1 to SEQ ID NO: 4 is schematically shown in Figure 2, the preferred method is as follows.

(1) extracting DNA;

(2) performing nested-PCR using a primer combination consisting of specific primers of SEQ ID NO: 1 to SEQ ID NO: 4 capable of detecting the Orientia Tsutsugamushi and the extracted DNA;

(3) Investigating the infection of Orientia Tsutsugamu through analysis of the PCR product.

In more detail, first, DNA is extracted from a sample such as blood according to a known method, and then the extract containing the extracted DNA is SEQ ID NO: 1 and SEQ ID NO. It is added to the primary PCR reaction solution containing 2 primer pairs. At this time, the primary PCR reaction solution should contain all the components necessary for PCR such as DNA polymerase (DNA polymerase) for PCR amplification. When the primary PCR is completed, a portion of the primary PCR product is added to the secondary PCR reaction solution containing the primer pairs of SEQ ID NO: 3 and SEQ ID NO: 4 of the specific primer combination of the present invention. At this time, the secondary PCR reaction solution should also contain all the components necessary for PCR including DNA polymerase for PCR amplification. Next, the reaction solution having undergone the nested-PCR process is electrophoresed in a conventional manner to determine whether or not Orientia Tsutsugamu is infected based on whether or not a specific band of Orientia Tsutsugamu is produced. In other words, when PCR products of 448 bp to 471 bp were identified, it indicated that the DNA sample prepared for the first extraction contained DNA of Orientia Tsutsugamu, which was the first sample used for DNA extraction, and infected with Orientia Tsutsugamu. Indicates that it was

Therefore, according to the present invention, it is possible to detect with high sensitivity and specificity whether the orientia Tsutsugamu infection is infected through nested-PCR using the specific primer combination of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these Examples.

Example 1: Preparation of Specific Primers

In the gene amplification of Orientia Tsutsugamu in the present invention, a region having a small difference by type of Orientia Tsutsugamu was used as an amplification range among gene regions encoding a 56 kDa type specific antigen (TSA) polypeptide.

Table 1 shows the nucleotide sequences of specific primers designed according to the present invention, and specific primers for gene amplification encoding 56 kDa type specific antigen (TSA) polypeptides of Orientia Tsutsugamu SEQ ID. The location is based on the Orientia Tsutsugamu Boryong type (NCBI accession number: AM494475). These primers have the complementary sequences inverted above, and the complementary sequences of the reversed in Table 1 are underlined.

division SEQ ID NO: Sequence Location (bp) 1st PCR
One 5'- GCA AT A TTG CTA GTG CAA TGT CTG C-3 ' 581,081-581,061 2 5'- ATG CA T GCA TGR CGC TKC AAT TTA-3 ' 579,935-579,953 2nd PCR
3 5'- ATA GG C CTA TAA GTA TWG CKG ATC G-3 ' 580,672-580,653 4 5'- CAT CT A GAY GCA CTA TTA GGC AAA-3 ' 580,211-580,229

Example 2: Sample Strain and DNA Preparation

Orientia Tsutsugamu strains include Orientia Tsutsugamu Kape (obtained from the Disease Control Center), Orientia Tsutsugamu Kato type (obtained from the Disease Control Center), Orientia Tsutsugamu Gili Cancer (obtained from the Disease Control Center), and Orientia Tsutsugamu Boryeong A mold (obtained from the Centers for Disease Control) was used. Control strains include Rickettia japonica (obtained from the Centers for Disease Control), mediate erythema fever , Rickettia sibinka (obtained from the Centers for Disease Control), and Rickettia akari (obtained from the Disease Control Center). ) And a rash-mediated strain, Rickettia typhi (obtained from the Centers for Disease Control) were used. DNA extraction was carried out using the Viral Gene-spin ^™ DNA / RNA Extraction Kit manufactured by Intron Biotechnology, Inc. according to the product usage instructions.

Example 3: Detection of Orientia Tsutsugamu

In order to confirm the effectiveness of the specific primers of the present invention and the detection method of Orientia Tsutsugamu using the same, the following experiment was performed. First, a PCR reaction solution was prepared with a total volume of 20 μl for the execution of the first PCR, and its components were composed of 2 μl of DNA extract prepared in Example 2 (about 50 ng of DNA) and 10 pmol / μl of SEQ ID NO: 1 To each specific primer of SEQ ID NO: 2, a PCR reaction solution containing all PCR necessary components, and distilled water. As the PCR reaction solution, one of the components, Maxime ^TM PCR PreMix (i-StarTaq) manufactured by Intron Biotechnology, Inc., was used, which was used according to the instructions for use of the product.

In the first step of the first PCR, the prepared PCR reaction solution was left at 94 ° C for 5 minutes to denature the DNA to be a template in PCR. Thereafter, a first PCR reaction was performed in a manner of repeating a total of 40 PCR cycles consisting of 30 seconds at 94 ° C, 30 seconds at 58 ° C, and 40 seconds at 72 ° C. After 40 repetitions, the PCR reaction solution was further left at 72 ° C. for 5 minutes to terminate the first PCR.

After the first PCR was completed, the second PCR was performed using the first PCR product as a template. The reaction solution was prepared with a total volume of 20 μl for the execution of the secondary PCR, and the components thereof were 1 μl and 10 pmol / μl of the first PCR product generated as a result of the first PCR. Each of the specific primers, PCR reaction solution containing all the necessary PCR components and distilled water. As the PCR reaction solution, one of the components, Maxime ^™ PCR PreMix (i-StarTaq), also manufactured by Intron Biotechnology, Inc., was used according to the instructions for use of the product.

In the first step of the second PCR, the DNA was first denatured by standing at 94 ° C for 5 minutes. Thereafter, a secondary PCR reaction was performed in a manner of repeating a total of 30 PCR cycles consisting of 30 seconds at 94 ° C, 30 seconds at 58 ° C, and 30 seconds at 72 ° C. After 30 repetitions, the PCR reaction solution was further left at 72 ° C. for 5 minutes to terminate the secondary PCR.

After the completion of the secondary PCR, the secondary PCR reaction solution was loaded onto a 1.5% agarose gel containing 0.1 μg / ml of RedSafe Nucleic Acid Staining Solution from Intron Biotechnology Co., Ltd. Youngdong was performed. After electrophoresis, PCR products were analyzed and the results were read.

The results of comparing the effects of the specific primers (primers with inverse complementary sequences) of the present invention with the general primers (primers without the reverse complementary sequences) are shown in FIG. 3. As can be seen from the results of Figure 3 it can be seen that the results are consistent with the immunological test results when following the present invention. For reference, it may be said that the method of the present invention is superior to the immunological method considering that it is much simpler and less time consuming than the immunological test. On the other hand, in the case of using the existing general primer, there were quite a few cases where it was not determined as positive among the positive samples. From this, it can be seen that the present invention is superior to the conventional PCR-based detection method.

Example 4: Measurement of sensitivity of PCR

In the PCR detection method using specific primers according to the present invention, the results of investigating the detection sensitivity of Orientia Tsutsugamu are shown in FIG. 4.

According to the results, it can be seen that the PCR detection method using the specific primers according to the present invention can provide a sufficient degree of sensitivity to be used to detect the infection of Orientia Tsutsugamu.

Example 5: Validation of PCR

The effectiveness of the present invention was investigated for various Orientia Tsutsugamu. As a result, the results showing that the detection method disclosed in the present invention works well for the Orientia Tsutsugamushi Born type, Orientia Tsutsugamshi Gill type, Orientia Tsutsugamshi Kato type, and Orientia Tsutsugamshi Kato type, are shown in FIG. It is.

In addition, as a result of examining the specificity of the PCR detection method using the specific primers according to the present invention, the bands of the characteristic amplification products could not be identified for the strains causing rash and erythema fever corresponding to the genus Rickettsia, such as Orientia Tsutsugashishi. The results are also shown in FIG. 5. Through this, it was confirmed that the present invention is specific to the identification of infection of Orientia Tsutsugamu, and can be effectively utilized.

Example 6: Validation in Clinical Samples

The results of PCR amplification of the randomly selected clinical sample using the method disclosed in the present invention are shown in FIG. 6, and the sample determined to be positive in FIG. 6 was subjected to conventional immunization against the first sample used for DNA extraction. All of the samples were found to be positive in the laboratory test. As a result, it could be seen that the present invention can be used as a clinical diagnosis method for the infection of Orientia Tsutsugamu.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a diagram showing a site amplified by primary PCR and secondary PCR in a gene encoding a 56 kDa type specific antigen (TSA) polypeptide of Orientia Tsutsugamu.

Figure 2 is a flow chart showing the orientia Tsutsugamu detection process using the PCR detection method according to the present invention.

Figure 3 is a result of comparing the detection method using the orientia Tsutsugamu detection method using the specific primers according to the present invention and the existing conventional primers. For reference, the results of immunological tests on the samples used for DNA extraction are also shown. In the immunological test results, "+" is positive and "-" is negative. Samples with the same number in each PCR detection experiment represent the same sample, and the sample with the same number in the immunological test is the test result of the corresponding sample used for extraction of the DNA used for PCR detection.

Figure 4 is an electrophoresis picture of the detection sensitivity for Orientia Tsutsugamu using specific primers according to the present invention. Lane M is a DNA size marker and lane N is a negative reference. 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg shown in the upper portion of the lanes represent the DNA concentrations extracted from Orientia Tsutsugamu used in each PCR reaction.

5 is a result of examining the effectiveness and specificity of the present invention, it is a result confirming the cross-reaction with the causative bacteria in the common pathogenic rickettsia genus. Lane M is DNA size marker, lane "1" is Orientia Tsutsugamu Boryeong, lane "2" is Orientia Tsutsugamu Giliam, lane "3" is Orientia Tsutsugamu Kato, lane "4" is Orientia Tsutsugamu Kaju Lanes 5 to 7 are causative agents of erythema fever, lane “5” is Rickettsia japonica, lane “6” is Rickettsia shivinca, lane “7” is Rickettsia acaria, and lane “8” is Rickettsia, the causative agent of rash. Typhi, lane N is a negative criterion, lane P is a positive criterion, PCR results using DNA extracted from a sample confirmed positive by conventional immunological methods.

Figure 6 shows the results of the present invention for any selected clinical sample. Lane M is a DNA size marker, lane N represents a negative criterion, and each number in the lane is a clinical sample number randomly assigned to the clinical sample.

<110> iNtRON Biotechnology, Co., Ltd. <120> Detection method of Orientina thstsugamushi using high Orientina          thstsugamushi-specific primers with specific structure of a          reverse complementary sequence <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 1st forward primer <400> 1 gcaatattgc tagtgcaatg tctgc 25 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 1st reverse primer <400> 2 atgcatgcat grcgctkcaa ttta 24 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 2nd forward primer <400> 3 ataggcctat aagtatwgck gatcg 25 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 2nd reverse primer <400> 4 catctagayg cactattagg caaa 24  

Claims (5)

A primer capable of amplifying a partial sequence of a gene encoding a 56 kDa type specific antigen polypeptide of Orientia tsutsugamushi . Primer for detecting orientia Tsutsugamu infection consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 4 characterized in that the complementary sequence of the reverse of the sequence is added. Kit for detecting the infection of Orientia Tsutsugamu, characterized in that it comprises the primer of claim 1. Orientia Tsutsugamushi related disease diagnostic kit comprising the primer of claim 1. Orientia Tsutsugamu infection test method using the primer of claim 1. The method of claim 4, wherein the inspection method, (1) extracting DNA from a sample; (2) performing nested-PCR using a primer combination consisting of SEQ ID NO: 1 to SEQ ID NO: 4 and the extracted DNA; (3) A method for testing the presence of Orientia Tsutsugamu infection, comprising the step of examining whether or not orientia Tsutsugamu infection through the analysis of the PCR product obtained from step (2).

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