JP2021516039A - Diagnosis method of scrub typhus using multi-copy gene - Google Patents

Abstract

The present invention relates to a method for diagnosing scrub typhus using a multicopy gene, preferably a multicopy gene of orientia tsutsugamushi for detecting Orientia tsutsugamoshi, which is the causative bacterium of scrub typhus. The present invention relates to a method for diagnosing scrub typhus by providing a primer pair based on the base sequence of Typhus and detecting Orientia tsutsugamushi using the primer pair. Since the diagnostic method using the primer pair of the present invention can detect with higher sensitivity and specificity than the conventional test method for detecting using a target gene, clinical diagnosis can be performed quickly and easily with high accuracy. ..

Description

The present invention relates to a method for diagnosing scrub typhus using a multicopy gene, and preferably amplifies the base sequence of the multicopy gene of Orientia tsutsugamushi, which is the causative bacterium of scrub typhus. It relates to a primer that can be used and a method for diagnosing scrub typhus using the primer.

Scrub typhus is transmitted by being bitten by larvae of hair mites infected by Orientia tsutsugamushi as an acute febrile illness, and exhibits characteristic clinical symptoms due to systemic vasculitis. The main host is rodents, and mites are hosts and vectors. Since it was first reported in Korea in 1951 as a major fall recessive disease, it has occurred throughout Korea between September and November, and in particular, 30% of patients who occur in the fall of Korea have scrub typhus. The disease is so common that it is diagnosed as.

Symptoms may be fever, chills, headache, muscle pain as initial symptoms after a latent period of 1 to 3 weeks after being bitten by an infected hair mite larva, and may be erythematous spots or papular. Rashes appear on the chest, abdomen, torso or upper and lower limbs, and in rare cases on the face, palms, and soles of the feet. Within a few days of onset, a black scab-covered eschar develops where the hair mite bites, and in most cases there are no symptoms such as pain or itching, and confirmation of the eschar is useful for rapid diagnosis. is there.

If the course of symptoms is not severe and responds well to antibiotic treatment, but the diagnosis is delayed, pneumonia, acute renal failure, meningitis, encephalitis, upper gastrointestinal bleeding, multi-organ dysfunction, and even myocardial infarction It may appear in the form of meningitis, and these complications can lead to death in some patients.

Therefore, rapid and accurate diagnosis is essential to improve the prognosis of patients.

Diagnosis of scrub typhus includes a method of culturing, separating and confirming orientia tsutsugamushi from the blood of a patient, and a method of detecting an antibody against orientia tsutsugamushi in the serum of a patient. However, the method of culturing the cells to diagnose the disease requires several weeks or more for the time required for culturing, and is not suitable for the purpose of diagnosing an actual patient.

The most commonly used test methods, which are mainly used for diagnosis, are indirect immunofluorescence antibody method (IFA), passive hemagglutination method (PHA) and immunochromatography (Immonochromatography).

Diagnostic methods using these antibodies have the drawbacks that they are often detected 1 to 2 weeks after the onset of symptoms, have very low sensitivity, have many false positives, and are not very useful for diagnosis.

For example, referring to FIG. 1, among the methods for diagnosing scrub typhus disclosed in the Disease Control Headquarters, the diagnosis using the indirect immunofluorescence antibody method (IFA) shows that when the IFA IgG antibody is 1: 256 or more, scrub typhus Judged as confirmed.

However, it may take several weeks for the antibody to rise and be diagnosed, and the sensitivity is relatively low at 46.7%, and 42.1% of all patients come to the hospital one week after the onset of symptoms. Only scrub typhus can be diagnosed, and there is a need for a new diagnostic method that is quicker and easier to determine.

As an alternative diagnostic method, a detection method based on PCR (Polymerase Chain reaction) has been proposed. PCR is a molecular biological method that can geometrically amplify the number of copies of the desired portion of DNA. Any part of DNA can be amplified by PCR as long as its sequence is known.

The PCR method is capable of conventional PCR performed by a conventional method, a nested PCR method that is 100 times more sensitive than the conventional PCR method by modifying it, and real-time monitoring of the PCR reaction. There is a real-time PCR method that allows the results to be confirmed quickly within 2 hours.

Conventionally, in the PCR diagnostic method for scrub typhus, PCR using various target protein genes (target protein genes) such as 46 kDa, 57 kDa, and groEL has been attempted, and nested PCR and real-time PCR are mainly used rather than conventional PCR. Was being done. Conventional PCR in the diagnosis of scrub typhus has the disadvantage that the sensitivity is very low within 10%, and in the case of nested PCR, PCR can be performed twice to improve sensitivity, but other PCRs. There is a drawback that the test time is further longer than that of the above, and a false positive rate is increased due to gene contamination because PCR is performed twice.

However, there is a method of diagnosing the test quickly by performing real-time PCR that monitors the PCR reaction in real time, but the fact is that it is difficult to use it universally in medical institutions because the testing equipment is very expensive. Is.

Therefore, it is urgent to develop a diagnostic method that is generally simple and low in cost while exhibiting high sensitivity and specificity.

The present invention has been devised to solve the above problems, and the present invention provides a method for diagnosing scrub typhus using a multicopy gene of Orientia tsutsugamushi. Is the main purpose.

Preferably, it is an object of the present invention to provide a method for diagnosing scrub typhus using a primer pair capable of amplifying the base sequence of a multicopy gene.

A second object of the present invention is to provide a primer pair containing a specific base sequence.

A third object of the present invention is to provide a PCR diagnostic kit containing a primer pair consisting of a specific base sequence.

Preferably, it is an object of the present invention to provide a PCT diagnostic kit capable of rapidly diagnosing scrub typhus by providing a primer pair consisting of a specific base sequence having excellent sensitivity and specificity. ..

In order to achieve the first object described above, the present invention discloses a primer containing a specific base sequence capable of amplifying the base sequence of the multicopy gene of Orientia tsutsugamushi. The primer of the present invention contains one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to SEQ ID NO: 36.

Preferably, the primer pair of the present invention contains two or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to SEQ ID NO: 36.

More preferably, the primer pair can contain the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In order to achieve the second object of the present invention, the method for diagnosing scrub typhus of the present invention can be detected using a primer pair capable of amplifying the base sequence of a multicopy gene.

The above diagnostic methods include (a) a step of separating DNA from a sample, (b) a step of performing PCR using the separated DNA as a template and using a primer pair of SEQ ID NO: 1 to SEQ ID NO: 36, and (c). The step of separating the PCR product can be included.

In order to achieve the third object of the present invention, the PCR kit for diagnosing scrub typhus can include a primer pair selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to SEQ ID NO: 36.

The primer pair containing a specific base sequence according to the present invention has high specificity for a multicopy gene.

In addition, the method for diagnosing scrub typhus according to the present invention can be detected with high sensitivity and specificity by PCR reaction using a primer pair containing a specific base sequence, so that clinical practice can be performed quickly and easily with high reliability. Can be diagnosed.

FIG. 1 is a graph of detection sensitivity by a diagnostic method using an indirect immunofluorescent antibody method. FIG. 2 is a flowchart showing a method for diagnosing scrub typhus using the PCR detection method according to the present invention.

Hereinafter, the present invention will be described in detail.

The method for diagnosing scrub typhus of the present invention can be detected using a primer pair capable of amplifying the base sequence of the multicopy gene of Orientia tsutsugamushi.

The multicopy gene refers to a gene that repeatedly exists at a plurality of positions in the Orientia tsutsugamushi gene, and preferably can contain the base sequences of 14 types of Tra-linked genes, and more preferably traB. , TraE, traC and TchA genes can be included.

The multicopy gene is a PCR target gene (Target gene).

The target protein genes such as 47 kDa, 56 kDa, and groEl-STG used in the conventional diagnostic method are present only at a certain position inside the whole gene, whereas the multicopy gene is repeated at various positions. Since it is present, the PCR efficiency is increased when PCR is performed to amplify the base sequence, so that the accuracy of diagnosis of scrub typhus can be improved.

The primer for diagnosing scrub typhus of the present invention contains one or more base sequences selected from the group consisting of the base sequences of SEQ ID NO: 1 to SEQ ID NO: 36.

In addition, the primer pair for diagnosing scrub typhus of the present invention contains two or more kinds of base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to SEQ ID NO: 36.

The primer pair can include the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Usually, the primer pair refers to two primers that do not have complementary base sequences.

Primers are short single-line oligonucleotides that are complementary to a specific gene sequence and serve as a starting point for DNA synthesis, and are used for PCR tests, DNA sequencing, and the like. Primers are also the most important factor in PCR testing and determine accuracy. The primer was designed based on the nucleotide sequence of the multicopy gene of the Boryon strain amplified from the blood of a patient infected with the Orientia tsutsugamushi strain and complemented with the multicopy gene. Has a typical base sequence.

The method for diagnosing scrub typhus is as follows: (a) a step of separating DNA from a sample, and (b) a step of performing PCR using the separated DNA as a template using the primer pairs of SEQ ID NO: 1 to SEQ ID NO: 36. And (c) the step of separating the PCR product can be included.

The DNA extraction in step (a) can be carried out by a method commonly used in the art and can be carried out using a commercially available DNA extraction kit.

In the PCR in the step (b), a PCR reaction can be carried out by synthesizing a complementary DNA strand (DNA strand) using the separated DNA as a template and the primer pair of the present invention using a DNA polymerizing enzyme.

In addition, a real-time PCR reaction can be carried out by further including a probe (Probe) based on the base sequence in the product amplified by the pair of primers in the step (b).

The probe means a nucleic acid fragment consisting of several to several hundred bases capable of specifically binding to a base sequence, and is labeled so that it can be confirmed whether or not a specific nucleic acid is present. it can. The probe can preferably be labeled with a fluorescent substance at the 5'-end and the 3'-end, respectively.

Further, more preferably, the fluorescent substance labeled at the 5'-terminal is 6-carboxyfluorescein (6-carboxyfluorescein, FAM), hexachloro-6-carboxyfluorescein (hexachloro-6-carboxyfluorescein, HEX), tetrachloro-. 6-carboxyfluorescein (tetraculo-6-carboxyfluorescein), and Cyanine-5 (Cy5), 6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein (6-Carboxy-4', 5'-Dichloro-2) ', 7'-Dimethoxyfluorescein, 6-JOE), tetrachlorofluorescein (TET), tetramethylrhodamine isothiacyanate, Texas Red, 5-carboxy-xmin- The fluorescent substance selected from the group consisting of ROX) and 6-carboxytetramethyl-rhodamine (6-carboxytetramethyl-rhodamine, TAMRA) and labeled at the 3'-end is 6-carboxytetramethyl-rhodamine (6-carboxytetramethyl). -Rhodamine, TAMRA) or black fluorescein-1,2,3 (BHQ-1,2,3), but not limited to.

The PCR is an abbreviation for polymerase chain reaction, which is a technique for amplifying a specific region of DNA or RNA in a large amount in vitro, and is a PCR reaction containing a plurality of components known in the art. It can be done using a mixture. In addition to the PCR primer pair provided in the present invention, the PCR reaction mixture can contain an appropriate amount of DNA polymerizing enzyme, dNTP, PCR buffer solution and water (dH ₂ O).

In addition, the PCR buffer solution can contain Tris-HCl, MgCl ₂ , KCl, and the like.

PCR usually consists of three reaction stages. The first is the DNA denaturation step, where the double-stranded DNA is separated into single-stranded DNA by applying temperature, and the second is the primer binding step, where the single-stranded DNA is denatured. When the temperature is lowered after coexisting with the primer, the primer pair is bound to the complementary single-stranded template DNA, and the third is the extension reaction in which four types of substrates (dNTPs) coexist. It consists of a stage in which a DNA polymerizing enzyme is allowed to act in the state to extend the primer.

As the PCR, a conventional PCR reaction can be used, and preferably, the most common conventional PCR, duplex PCR and nested PCR in which two PCRs are carried out simultaneously or continuously, Any of the expensive real-time PCRs that can confirm the results in real time can be used, and most preferably conventional PCR or real-time PCR can be used.

Since the real-time PCR can monitor and confirm the increase in PCR amplification products in real time, it is not necessary to use an additional electrophoretic analysis method, and PCR amplification is performed at the endpoint (End point). Compared with the existing PCR method for confirming the product, PCR can be analyzed quickly and easily, and the risk of contamination is low.

The method for detecting a real-time PCR amplification product is specific to a target sequence, and a probe to which a fluorescent dye (die) is bound is used to distinguish even similar sequences with high detection specificity. Can be detected.

Further, in the step (c), the PCR product can be separated by a method widely known in the art. Preferably, it can be confirmed by agarose gel or polyacrylamide gel electrophoresis or fluorescence analyzer (ABI prism 3100 genetic analyzer-elechromegram). After electrophoresis, the results of electrophoresis can be analyzed by ethidium bromide staining.

The PCR kit for diagnosing scrub typhus of the present invention can contain a primer pair selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to SEQ ID NO: 36.

In addition to the primer pair capable of amplifying the base sequence of the multicopy gene, the detection PCR kit can include an enzyme involved in the polymerization enzyme chain polymerization reaction, dNTP, and a buffer solution, and PCR can be used. It can contain materials for PCR such as mineral oil, a standard marker, and a staining reagent, bromophenol blue or xylene FF, which are components necessary for performing electrophoresis, which can confirm whether or not the product is amplified.

Hereinafter, the present invention will be described in more detail with respect to a method for detecting and diagnosing bacteria according to the following examples.

The following examples are merely for the purpose of explaining the present invention in detail, and it is obvious to a person having ordinary knowledge in the art that the content and scope of the present invention are not limited by the examples. Is.

<Example>
1. 1. Primer Preparation Designed based on a multicopy gene amplified from the blood of patients infected with the Boryeong strain, which is common in Korea.

More preferably, the primer is Genbank Accession No. AM494475.1: O.D. It was prepared with a primer (position 14-34) in the forward direction and a primer (position 171-151) in the reverse direction with respect to the tsutsugamushi Boryoung complex genome thcA gene (conserved hypothetical protein), and sequence numbers 1 to 36, respectively.

The probe was prepared by Taqman probe (position 58-78) and is represented by SEQ ID NOs: 37 and 38.

The primers and probes prepared according to the examples of the present invention are shown in Table 1 below.

The primer can have a base sequence complementary to the multicopy genes tchA, traB, traE, and trbC.

The primer sequences had a length of 19 nt to 30 nt, a GC content of 20 to 60%, and a Tm value of 50 to 70 ° C.

2. Specimen preparation For patients who visited Chosun University Hospital between 2007 and 2008, there was crust or hemispherical true rash at that time, headache, general fragility, muscle pain, cough, nausea, abdominal pain, etc. Blood samples of patients with two or more types of clinical symptoms were taken, and genes were extracted and used for testing.

A definitive diagnosis of scrub typhus is defined as a 4-fold or higher increase in lgG antibody against orientia scrub typhus by indirect immunofluorescence test (IFA) in blood samples, and the blood of 52 patients with a definitive diagnosis of scrub typhus is positive. As a control group, the blood of 20 patients diagnosed with other diseases other than scrub typhus was used as a negative control group.

3. 3. Detection of Orientia Tsutsugamushi DNA was detected in the positive control group and negative control group samples prepared to confirm the effectiveness of the prepared primers and diagnostic method, and PCR was performed as shown in FIG. ..

Whole blood was collected from the patient, the leukocyte soft layer was separated, and then the DNA was separated using the QIA amp DNA mini-kit (Qiagen, Germany). The separation method was carried out according to the manual included in the kit, and the separated DNA was used as a template for the detection of Orientia tsutsugamushi.

Conventional PCR, nested PCR and real-time PCR were performed using the extracted DNA as a template.

At this time, as the primers used in the experiment, the primers having the nucleotide sequences of SEQ ID NOs: 1 to 36 of the present invention and the probes having the nucleotide sequences of SEQ ID NOs: 37 and 38 were used.

Conventional PCR and nested PCR are ^{2 μl template DNA in AccuPower TM} PCR PreMix (1 U Top polymerase, 250 μm dNTP, 10 mM Tris-HCl (pH 9.0; Bioneer)), 1 μl each of 10 pmole / μl forward and reverse primers, 16 μl. After preparing a total of 20 μl of the reaction solution by adding the sterilized tertiary distilled water of the above, the tubes were mixed ^{well and set in the Biosystems Veriti TM} 96-well Primer cycle (Applied Biosystems), and then PCR was performed.

For real-time PCR, prepare a reaction solution containing 1 μl of 2 pmole / μl probe in the same manner as described above, and then mix the tubes well to mix the BIONEER Exiciller ^TM 96. After setting in Real-Time Quantitative Thermal Block (Applied BIONNER), PCR was performed, and FAM (6-carboxyFluo-rescein) and BHQ-1 (Black Hole Quench-1) at the 5'and 3'-ends of the probe were used. And used.

The PCR execution conditions are shown in Table 2.

After completion of PCR, 1.5% agarose gel (Seachem LE agarose, Cambrex BioScience) containing 0.5 ng / ml EtBr (ethidium bromide, Bioneer) was loaded on a Bionia electrophoresis apparatus (Bioneer), and 100 , Bioneer), the PCR amplification product was electrophoresed for 40 minutes, and the results were observed.

As a result of the PCR test, no band was observed in the PCR amplification product for the negative control group when electrophoresed. In addition, the size of the PCR amplification product by the primer pair of the present invention is shown in Table 3, and it was decided to confirm whether orientia tsutsugamushi was detected by confirming the band of that size. It was confirmed that the PCR amplification product yielded results consistent with the indirect immunofluorescence test method.

<Comparison example>
1. 1. Detection of Orientia Tsutsugamushi by Conventional Method In order to compare the effectiveness of the primers and diagnostic methods of the present invention, a comparative experiment was conducted as follows.

As the template DNA, the one isolated from the blood sample was used in the same manner, and in each experiment, conventional PCR and nested PCR for 56 kDa and 47 kDa, which are target protein genes commonly used for the detection of scrub typhus, were performed. The results are shown in Table 4.

The primer pairs used for each PCR were prepared based on the references or directly designed and used, and PCR was carried out in the same manner as in Example 3.

Primer pairs for amplifying the 47kDa gene, references (Jiang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, Richards AL.Development of a quantitative realtime polymerase chain reaction assay specific for Orientia tsutsugamushi.Am J Trop Med It was prepared with reference to Hyg. 2004 Apr; 70 (4): 351-6.).

In addition, the primer pair for amplifying the 56 kDa gene can be found in the references (Kim DM, Yun NR, Yang TY, Lee JH, Yang JT, Sim SK, et al. Usefulness of guided PCR for the digital diagnosis). : Aprospective study. Am J Trop Med Hyg. 2006 Sep; 75 (3): 542-5.).

<Results> Confirmation of effectiveness of method for diagnosing scrub typhus In the methods for detecting orientia tsutsugamushi in Example 3 and Comparative Examples using the primers of the present invention, the detection sensitivity and specificity of orientia tsutsugamushi (sensitivity) and specificity ( Table 5 shows the results of measuring the specificity) and the detection time.

As can be seen from Table 5, when the conventional (C) -PCR targeting the TraB gene of Example 3 was performed, the time was shorter than that of the nested (N) -PCR targeting the 56 kDa and 47 kDa genes of Comparative Example 1. Was required.

Moreover, when conventional (C) -PCR was performed targeting the TraB1 and TraB2 genes of Example 3, the sensitivities were 82.7% and 88.5%, respectively.

In particular, when the Tch gene is used as a target, the sensitivity is 90.4% when conventional PCR (C-PCR) is performed, and the detection time is 1 when real-time PCR (Q-PCR) is performed. The sensitivity was 98.1% even though it was only ~ 2 hours.

Therefore, as shown in Table 5 below, the primer pair of the present invention achieved the purpose and effect that the present invention aimed to achieve.

From the above results, it can be confirmed that the PCR detection method using the primer pair according to the present invention can provide sufficient sensitivity to confirm the presence or absence of infection with Orientia tsutsugamushi.

As described above, a specific part of the content of the present invention is described in detail, and for a person having ordinary knowledge in the art, such a specific description is merely an example of accurate implementation. This does not limit the scope of the invention, and it can be said that the substantial scope of the invention is defined by the appended claims.

Claims (7)

A method for diagnosing scrub typhus, which comprises using a multicopy gene of Orientia tsutsugamushi. The diagnostic method for scrub typhus according to claim 1, wherein the detection is performed using a primer pair capable of amplifying the base sequence of the multicopy gene. A primer for diagnosing scrub typhus, which comprises one or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to SEQ ID NO: 36. A diagnostic primer pair for scrub typhus, which comprises two or more base sequences selected from the group consisting of the base sequences of SEQ ID NOs: 1 to SEQ ID NO: 36. The primer pair for diagnosing scrub typhus according to claim 4, wherein the primer pair contains the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. (A) Step of separating DNA from the sample;
(B) A step of performing PCR using the separated DNA as a template and a primer pair of SEQ ID NO: 1 to SEQ ID NO: 36; and (c) a step of separating the PCR product, Tsutsugamushi disease. Diagnostic method. A PCR kit for diagnosing scrub typhus, which comprises a primer pair selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to SEQ ID NO: 36.

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