WO2011162441A1

WO2011162441A1 - Primer pairs for detecting orientia tsutsugamushi and detection method using the same

Info

Publication number: WO2011162441A1
Application number: PCT/KR2010/005765
Authority: WO
Inventors: Dong Min Kim
Original assignee: Industry-Academic Cooperation Foundation, Chosun University
Priority date: 2010-06-24
Filing date: 2010-08-27
Publication date: 2011-12-29
Also published as: KR101185117B1; CN103080312A; KR20110139905A; HK1184825A1; CN103080312B

Abstract

The present invention relates to a diagnosis method of scrub typhus, more precisely, to a diagnosis method of scrub typhus by detecting Orientia tsutsugamushi, the cause of scrub typhus, comprising the following steps: preparing primer pairs based on the 16s ribosomal gene sequence of Orientia tsutsugamushi; and detecting Orientia tsutsugamushi using the prepared primer pairs. The diagnosis method of the present invention not only demonstrates higher diagnostic accuracy than other conventional methods amplifying and analyzing 47kDa, 57kDa, and groEL, but also is very simple and easy,suggesting that it is excellent in the aspect of cost-effect.

Description

PRIMER PAIRS FOR DETECTING ORIENTIA TSUTSUGAMUSHI AND DETECTION METHOD USING THE SAME

The present invention relates to primer pairs for diagnosing Orientia tsutsugamushi and a diagnosis method of Orientia tsutsugamushi using the same, more specifically, to primer pairs that are capable of amplifying 16s ribosomal RNA gene base sequence of Orientia tsutsugamushi, the cause of scrub typhus, and a detection method of scrub typhus by detecting Orientia tsutsugamushi using the same.

Scrub typhus is an acute febrile disease, which is transmitted via chigger infected with Orientia tsutsugamush, and shows characteristic clinical symptoms due to systemic vasculitis. A rodent is a major host and a mite(chigger) is a host and vector.

Since scrub typhus was first reported in Korea in 1951, this major autumn febrile disease has been spread all over the country particularly in between September and November. Particularly, so many cases have been occurring as 30% of all patients in fall are diagnosed as scrub typhus.

Symptoms such as fever, chill, headache, muscle ache, and erythematous papular rash would be developed in 1-2 weeks after the chigger bite. A detection of the developed characteristic eschar would be helpful for early diagnosis of the disease. Generally, the progress of this disease is not serious and an antibiotic works well for the disease. However, in case of a late diagnosis, complications in the form of pneumonia, acute renal failure, encephalomeningitis, encephalitis, upper gastrointestinal bleeding, multiple organ dysfunction, or even worse myocardial infarction or stroke can be developed. These complications might be lethal, so that rapid and accurate diagnosis is essential for the improvement of prognosis of a patient.

Up to date, the methods for diagnosing scrub typhus are methods such as by confirming Orientia tsutsugamushi by culturing, by detecting the antibody against Orientia tsutsugamushi in the serum of a patient and by examining DNA of Orientia tsutsugamushi separated from blood of a patient by PCR (polymerase Chain Reaction) amplification.

Among them, in the diagnosis method by culturing Orientia tsutsugamushi, it takes at least several weeks to culture Orientia tsutsugamushi, suggesting that this method is practically improper for the purpose of diagnosis of a patient. So, the most common diagnosis methods are indirect fluorescent antibody technique and immuno enzyme technique, which are serological test methods.

However, the above methods still have disadvantages of false positive resulting from the cross-reaction with other pathogenic microorganisms, low sensitivity in case of test in an early stage of the disease due to requiring several days until an antibody is formed after symptoms of the disease begins, and additional tracking tests required for comfirming diagnosis.

Another diagnosis method of scrub typhus, which is a method of detecting Orientia tsutsugamushi specific DNA from blood of a patient by PCR in order to make the rapid diagnosis of scrub typhus in its early stage, has been introduced. Examples of PCR comprise the conventional PCR, or nested PCR obtained by the modification of the conventional PCR and showing 100 times greater sensitivity than the conventional PCR, or real time PCR during which the results can be confirmed rapidly within two hours.

In the conventional PCR diagnosis of scrub typhus, PCR using various target protein genes such as 46kDa, 57kDa, and groEL, etc, has been tried, and nested PCR or real time PCR has been more preferred over the conventional PCR. Because when the conventional PCR was performed by using 46kDa or 57kDa gene as a target, sensitivity was as low as at best 10%. However, nested PCR has a drawback that it does take longer time to test than other PCR, with PCR performed twice to increase sensitivity. Also, real time PCR has a drawback that with very expensive testing equipments required, there was a limitation in use universally in most medical institutes.

Therefore, it is urgently requested to develop a novel diagnosis method which is mostly simple and low-cost as well as presenting high sensitivity and specificity.

The present inventors tried to develop a novel method to detect Orientia tsutsugamushi with ease and simplicity from the blood sample of a patient. As a result, the present inventors completed this invention by confirming that even with the existing conventional PCR, highly accurate diagnosis could be achieved by amplifying specifically a part of 16s ribosomal RNA gene sequence of Orientia tsutsugamushi.

Therefore, the present invention provides a detection method of Orientia tsutsugamushi by amplifying 16s ribosomal RNA gene of Orientia tsutsugamushi using the conventional PCR.

The novel method of the present invention overcome a problem that the sensitivity in the conventional PCR is within 10%, and also overcomes the problems that in nested PCR, performing PCR twice or more leads to more labors and time than needed and higher potential possibility of contamination, and the problem that in real-time PCR, expensive equipments are required. Therefore, the method of the present invention is an excellent method in cost-labor efficient aspects, which also provides diagnostic accuracy comparable to that of the PCRs.

To achieve the above objects, the present inventors designed a detection method of Orientia tsutsugamushi to amplify and identify 16s ribosomal RNA gene sequence of Orientia tsutsugamushi. This method can be achieved by preparing primers that can specifically amplify 16s ribosomal RNA of Orientia tsutsugamushi.

The preparation of the primers were designed based on the 16s ribosomal gene base sequence amplified from the blood sample of a patient infected with Orientia tsutsugamushi Boryoung strain, which was very common in Korea.

Thus, it is an object of the present invention to provide a method for diagnosis of scrub typhus and a method for detecting Orientia tsutsugamushi characterized by comprising the step of amplifying a target protein gene using the primers for 16s ribosomal RNA in blood sample of a patient suspicious with scrub typhus.

It is another object of the present invention to provide a PCR kit for the detection of Orientia tsutsugamushi containing the primers.

The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments and claims.

The present invention relates to a method for diagnosis of scrub typhus, more specifically, to primers that are capable of amplifying the 16s ribosomal RNA gene base sequence of Orientia tsutsugamushi, the cause of scrub typhus, and a method for diagnosis and detecting Orientia tsutsugamushi using the same.

The diagnosis method of the present invention presents excellent and high diagnostic accuracy, compared with the conventional methods analyzing by amplifying 46kDa, 57kDA and groEL genes. Also, when the primers of the present invention are applied to the conventional PCR, comparatively improved diagnostic accuracy is obtained. Moreover, the method of the invention is not only superior in diagnostic accuracy to nested PCR or real time PCR but also simple and inexpensive.

Fig. 1 is graphs illustrating the comparison of diagnostic efficiency of the detection method of Orientia tsutsugamushi using the primer pair composed of the sequences represented by SEQ. ID. NO. 1 and NO. 2 originated from 16s ribosomal RNA of the present inventionto that of the conventional detection methods of comparative examples 1 - 6, with receiver operator characteristic (ROC) curve.

The present invention relates to a diagnosis method of scrub typhus. More precisely, the invention relates to a detection method of Orientia tsutsugamushi using primers for PCR to amplify the 16s ribosomal RNA gene base sequence of Orientia tsutsugamushi, the cause of scrub typhus.

The present invention also provides primers for PCR to amplify the 16s ribosomal RNA gene base sequence of Orientia tsutsugamushi, the cause of scrub typhus.

The present invention also provides a PCR kit for the detection of Orientia tsutsugamushi containing the primers.

Hereinafter, the present invention is described in detail.

The present invention is characterized by preparing the primer pairs for PCR to amplify 16s ribosomal RNA gene of Orientia tsutsugamushi and detecting the amplified 16s ribosomal RNA gene using the primers.

The primers of the present invention were designed based on the 16s ribosomal gene base sequence amplified from the blood sample of a patient infected with Orientia tsutsugamushi Boryoung strain, which was very common in Korea. The primers also could detect other prototype strains such as Gilliam, Karp, Kato, and various other strains.

The present invention is also characterized by amplifying the target protein gene, which is 16s ribosomal RNA gene in the blood sample from a suspected case of scrub typhus by using the primers.

The PCR is an abbreviation for polymerase chain reaction. The PCR comprises all the PCR of the most common conventional PCR, duplex PCR and nested PCR performing PCR twice consecutively, and high-cost real time PCR during which the results can be confirmed in real time, and most preferably it comprises the conventional PCR.

The PCR kit for detection herein may include materials for PCR such as the enzyme involved in polymerization, dNTP, buffer, mineral oil, standard marker, the staining reagent bromophenol blue or xylene FF, as well as the primer pairs for PCR for the amplification of 16s ribosomal RNA gene of Orientia Tsutsugamushi.

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Primer preparation

Primer pairs were designed based on the 16s ribosomal gene base sequence amplified in the blood sample from a patient infected with tsutsugamushi Boryoung strain, which was very common in Korea, but could detect other prototype strains such as Gilliam, Karp, Kato, and various other strains. More specifically, the primer pair was prepared as a forward primer (position 403-428) and a reverse primer (position 577-601) for GenBank Accession No. bankit1356164 HM352765: O. tsutsugamushi Boryoung Genotype, which were represented by SEQ. ID. NO. 1 and NO. 2, respectively. Forward and reverse primers were also prepared for other regions, which were represented by SEQ. ID. NO 3 - NO. 36 (see Table 1), respectively.

Table 1

Example 2: Sample preparation and vital statistics

Among patients at the age of 18 or above who went to a hospital because of acute febrile disease in 4 weeks from the outbreak, from 2007 to 2008, those who showed at least two symptoms selected from the group consisting of eschar, erythematous papular rash, headache, general prostration, muscle ache, cough, nausea, and stomachache were selected for blood sampling.

In the blood sample where IFA titer for Orientia tsutsugamushi was increased at least 4 times, the patient of that sample was diagnosed as scrub typhus(tsutsugamushi). So, such patients were grouped into tsutsugamushi patient group (scrub typhus group). In patients in the febrile control group (non-scrub typhus group), indirect fluorescent antibody against Orientia tsutsugamushi was not detected in their blood, and instead they were diagnosed as another febrile disease except scrub typhus by the tests such as serology, culture, and peripheral blood smear tests, etc, by Infectious diseases physicians.

An experiment was performed with blood samples of randomly selected 115 patients diagnosed as scrub typhus and 52 patients confirmed as febrile control group (non-scrub typhus). Vital statistics were also calculated and the results are shown in Table 2.

Table 2

As shown in Table 2, in the aspect of the average age of all patients (patient group: 66, control group: 53, mean value: 69 and 54 respectively), the average age of the tsutsugamushi patient group (scrub typhus group) was meaningfully higher than that of the febrile control group (non-scrub typhus group) (p=0.0002). There were no statistically meaningful differences between the gender ratio in the tsutsugamushi patient group (scrub typhus group) and that in the febrile control group (non-scrub typhus group) (p=0.693) (Table 2).

Example 3: PCR diagnosis using the primer pairs of the present invention

To confirm the efficacy of the primer pairs prepared and the comparative examination method, DNA was extracted from the blood samples of tsutsugamushi patients prepared in example 2. Particularly, whole blood was taken from a patient. Buffy coat was separated. Then, DNA was separated by using QIA amp DNA mini kit (Qiagen, Germany). The separation was performed by following the protocol enclosed in the manual. The separated DNA was used as the template for detecting Orientia tsutsugamushi.

As a method for PCR diagnosis, the conventional PCR was selected herein and the extracted DNA was used as the template. For the PCR, 20 ㎕ of reaction mixture was prepared by adding 2 ㎕ of template DNA, 1 ㎕ of 5 pmol/㎕ forward primer and 1 ㎕ of 5 pmol/㎕ reverse primer, and 16 ㎕ of sterilized deionized water to AccuPowerTM PCR PreMix (1 U Top polymerase, 250 uM dNTP, 10 mM Tris-HCl (pH9.0); Bioneer, Daejeon, Korea). The tube containing the reaction mixture was well stirred, which was set in Biosystems VeritiTM 96-well Thermal cycler (Applied Biosystems, Foster City, CA), followed by PCR. PCR conditions are summarized in Table 3.

Upon completion of PCR, to confirm PCR final product, 1.5% agarose gel (Seakem^R LE agarose; Cambrex Bio Science, Rockland, ME) containing 0.5 ng/mL Etbr (Ethidium bromide; Bioneer, Daejeon, Korea) was loaded in Bioneer electrophoresis machine (Bioneer, Korea), followed by electrophoresis at 100V (1X TAE; Bioneer, Korea) for 40 minutes.

The primers used in this experiment were the primer pair composed of sequences represented by SEQ. ID. NO. 1 and NO. 2, respectively. The amplified product of 16s ribosomal RNA gene by PCR of the invention is expected to have a size of 199 bp. The detection of a band of corresponding size confirmed whether Orientia tsutsugamushi was detected or not.

Table 3

Comparative Examples 1~6:

To compare the diagnostic accuracy of the detection method using the primer pairs of the present invention with others, the following comparative experiments were performed. At this time, the template DNA was the same as that prepared in example 3.

The conventional PCR (47kDa C-PCR: comparative example 1), nested PCR (47kDa N-PCR: comparative example 2) and real time PCR (47kDa Q-PCR: comparative example 3) targeting 47kDa generally used for detecting tsutsugamushi were performed. The conventional PCR (56kDa C-PCR: comparative example 4) and nested PCR (56kDa N-PCR: comparative example 5) targeting 56kDa and the conventional PCR targeting groEL (groEL C-PCR: comparative example 6) were also performed. The efficacies of those methods were compared.

The primers for each PCR were prepared according to the references stated in Table 4 or designed by the inventors, and the method is explained hereinafter.

Table 4

1) Conventional PCR (C-PCR)

A total 20㎕ of reaction mixture was prepared by mixing 2 ㎕ of DNA, 1 ㎕ of forward primer, 1 ㎕ of reverse primer, AccuPowerTM PCR PreMix (1 U Top polymerase, 250uM dNTP, 10 mM Tris-HCl (pH9.0), 30 mM KCl, 1.5mM MgCl₂, stabilizer and tracking dye; Bioneer, Daejeon, Korea) and sterilized deionized water.

At this time, ach PCR was performed with each primer as shown in Table 4 by using Biosystems VeritiTM 96-well Thermal cycler (Applied Biosystems, Foster City, CA). PCR conditions for each target gene are summarized in Table 5.

To confirm PCR product, 1.5% agarose gel (Seakem^R LE agarose; Cambrex Bio Science, Rockland, ME) containing 0.5 ng/mL Etbr (Ethidium bromide; Bioneer, Daejeon, Korea) was loaded in Bioneer electrophoresis machine (Bioneer, Korea), followed by electrophoresis at 100V (1X TAE; Bioneer, Korea) for 40 minutes.

[Corrected under Rule 26 14.09.2010]
Table 5

2) Nested PCR (N-PCR)

For the PCR targeting 56kDa gene, P10 and P11 (5 pmoles/㎕) were used as internal primers and P34 and P55 (5 pmole/㎕) were used as external primers. The first PCR reaction mixture was prepared by the same manner as used for the preparation of the conventional PCR. The second round PCR was performed using the first PCR product (1㎕/ 20㎕ total volume) as template DNA with the primers P10 and P11 (5 pmoles/㎕). The final PCR product was electrophoresed on 1.5% agarose gel at 100 V. PCR targeting 47 kDa gene was performed by the same manner as described in 56kDa N-PCR except that different primers were used.

3) Real time PCR (Q-PCR)

Real time PCR targeting 47kDa gene was performed with 20 ㎕ of reaction mixture (5 ㎕ of genomic DNA, 1 ㎕ of 5 pmoles/㎕ forward primer and 1 ㎕ of 5 pmoles/㎕ reverse primer (OtsuFP630 and OtsuRP747), 1 ㎕ of 2 pmoles/㎕ probe (OtsuPR665), 4 ㎕ of master mix (reaction buffer, FastStart Taq DNA polymerase, MgCl₂, dNTP (dUTP instead of dTTP)), and distilled water).

The real-time PCR conditions were as follows: at 95℃ for 10 minutes, two steps at 95℃ for 10 seconds each, at 60℃ for 30 seconds (45 cycles) and finally at 40℃ for 30 seconds. The result was analyzed by Light Cycler software 4.0 program.

5' and 3' ends of the probe OtsuPR665 were labeled with FAM (6-carboxyFluo-rescein) and BHQ-1 (Black Hole Quencher-1).

Example 4: Comparative analysis of diagnostic accuracy

Diagnostic accuracy of the detection method of Orientia tsutsugamushi using the primer pair composed of sequences represented by SEQ. ID. NO. 1 and NO. 2 originated from 16s ribosomal RNA gene of the present invention and other detection methods were evaluated.

For the evaluation, sensitivity and specificity were presented as receiver operator characteristic (ROC) curve concept. The diagnostic accuracy was compared with those of other detection methods by using MEDCALC software program (MedCalc Software, Mariakerke, Belgium, version 11.2.1), and the results are shown in Table 6, Fig. 1 and the below <result> (Table 6, Fig. 1).

Diagnostic accuracy of the PCR method for tsutsugamushi of the present invention

PCR was performed with the blood samples of 115 scrub typhus patients and 52 patients diagnosed as other diseases (non-scrub typhus group). From the result of 47kDa C-PCR, it was confirmed that 3 of the 115 patients (3%) were positive. From the result of 47dKa N-PCR, it was confirmed that 93 of the 115 patients were positive, indicating the sensitivity was 81%.

In 47kDa Q-PCR, 38 cp or above was set as negative, and under that condition, 87 of the 115 scrub typhus patients were positive (76% of positive rate).

In C-PCR targeting 56kDa protein gene, 9 of the 115 scrub typhus patients were positive, and 92 of the 115 patients were positive in N-PCR (80% of sensitivity).

In C-PCR targeting groEL gene, 76 of the 115 scrub typhus patients were positive, suggesting 66% of sensitivity.

In the PCR method of the present invention targeting 16s ribosomal RNA gene (C-PCR of example 3), 100 of the 115 scrub typhus patients were positive, suggesting 86% of sensitivity.

In the non-scrub typhus group, no patients were positive in any PCR test except that 2 blood samples were confirmed as positive in 47kDa Q-PCR with the non-scrub typhus group.

Comparison of diagnostic accuracy of the detection method of the present invention to that of the detection methods of comparative examples

1) Comparison of diagnostic accuracy according to each target gene in the conventional PCR

AUC of the conventional PCR was as follows. AUC of 16s C-PCR was 0.94 (95% CI, 0.89-0.97), and AUC of 47kDa C-PCR was 0.51 (95% CI, 0.44-0.59). AUC of 56kDa C-PCR was 0.54 (95% CI, 0.46-0.62), and AUC of groEL C-PCR was 0.8 (95% CI, 0.77-0.88). AUC of 16s C-PCR has statistically meaningful differences over those of other C-PCRs (16s C-PCR vs 47kDa C-PCR, p<0.001; 16s C-PCR vs 56kDa C-PCR, p<0.001). AUC of 47kDa C-PCR had no statistically meaningful differences over that of 56kDa C-PCR (p=0.081) (Fig. 1).

2) Comparison of diagnostic accuracy according to PCR in each target protein gene

C-PCR targeting 47kDa gene demonstrated 3% (95% CI, 0.5-7.4) of sensitivity and 100% (95% CI, 93.2-100) of specificity. In the case of N-PCR targeting the gene, sensitivity was 81% (95% CI, 72.5-87.6) and specificity was 100% (95% CI, 93.2-100). Diagnostic accuracy of 47kDa Q-PCR was 76% (95% CI, 66.8-83.2) of sensitivity and 96% (95% CI, 86.8-99.5) of specificity when 38 cp was determined as cut-line for negative result. AUC of 47kDa N-PCR was 0.9 (95% CI, 0.85-0.94), and AUC of Q-PCR was 0.86 (95% CI, 0.8-0.91). AUC of C-PCR was 0.51 (95% CI, 0.44-0.59). There were statistically meaningful differences in each comparision (N-PCR vs C-PCR, p<0.001; N-PCR vs Q-PCR, p=0.012, C-PCR vs Q-PCR, p<0.001).

In C-PCR targeting 56kDa gene, sensitivity was 8% (95% CI, 3.6-14.3) and specificity was 100% (95% CI, 93.2-100). In N-PCR targeting 56kDa gene, sensitivity was 80% (95% CI, 71.5-86.9) and specificity was 100% (95% CI, 93.2 100). In the meantime, AUC (area under the curve) of N-PCR was 0.9 (95% CI, 0.46-0.61), and AUC of C-PCR was 0.54 (95% CI, 0.46-0.62), suggesting that there was a statistically meaningful difference (N-PCR vs C-PCR, p<0.001).

Diagnostic accuracy was compared according to AUC among 47kDa N-PCR and Q-PCR, 56kDa N-PCR, and the detection method of tsutsugamushi using the primers for 16s ribosomal RNA gene. As a result, C-PCR of the present invention using the primers for 16s ribosomal RNA gene demonstrated superior AUC values to any other methods and the statistically meaningful difference (16s C-PCR vs 47kDa N-PCR, p=0.017; 16s C-PCR vs 56 kDa N-PCR, p=0.03; 16s C-PCR vs 47kDa Q- PCR, p<0.001) (Fig. 2).

Table 6

That is to say, the comparison of diagnostic accuracy with AUC for tsutsugamushi shows that the diagnostic accuracy of the detection method of tsutsugamushi of the present invention (example 3) using the primers for 16s ribosomal RNA was superior to that of other conventional detection methods (comparative examples 1 6), and the difference in diagnostic accuracy was statistically meaningful. Moreover, it shows that the conventional PCR with the primers for 16s ribosomal RNA gene invented by the present inventors demonstrated much higher diagnostic accuracy than nested PCR and real-time PCR amplifying 47kDa gene which had been known to be very accurate and appropriate for disease diagnosis.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

1. Jiang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, Richards AL. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg. 2004 Apr;70(4):351-6.

2. Kim DM, Yun NR, Yang TY, Lee JH, Yang JT, Shim SK, et al. Usefulness of nested PCR for the diagnosis of scrub typhus in clinical practice: A prospective study. Am J Trop Med Hyg. 2006 Sep;75(3):542-5.

3. Park HS, Lee JH, Jeong EJ, Kim JE, Hong SJ, Park TK, Kim TY, Jang WJ, Park KH, Kim BJ, Kook YH, Lee SH. Rapid and simple identification of Orientia tsutsugamushi from other group rickettsiae by duplex PCR assay using groEL gene. Microbiol Immunol. 2005;49(6):545-9.

Claims

A primer pair for the diagnosis of scrub typhus composed of two sequences selected from the group consisting of:

oligonucleotide sequences represented by SEQ. ID. NO. 1 ~ NO. 36; and

sequences of at least 18 mer having at least 90% homology with said sequences.
The primer pair for the diagnosis of scrub typhus according to claim 1, composed of the sequences represented by SEQ. ID. NO. 1 and NO. 2.
A diagnosis method of scrub typhus using the primer pair of claim 1.
The diagnosis method of scrub typhus according to claim 3, wherein the primer pair composed of the sequences represented by SEQ. ID. NO. 1 and NO. 2.
The diagnosis method of scrub typhus according to claim 3 or claim 4, comprising detecting Orientia tsutsugamushi with polymerase chain reaction (PCR).
The diagnosis method of scrub typhus according to claim 3 or claim 4, wherein the detecting Orientia tsutsugamushi comprises the following steps of;

amplifying 16s rRNA gene sequence of Orientia tsutsugamushi or a part of the sequence; and

identifying thereof.
The diagnosis method according to claim 5, wherein the polymerase chain reaction (PCR) is the conventional PCR.
A PCR kit for detecting Orientia tsutsugamushi by the amplification of 16s rRNA gene sequence or a part of the sequence, comprising the primer pairs of claim 1 or 2.
A PCR kit for detecting Orientia tsutsugamushi by the amplification of 16s rRNA gene sequence or a part of the sequence.
A PCR kit for detecting Orientia tsutsugamushi by amplification of 16s rRNA gene sequence or a part of the sequence, comprising primer pair composed of the sequences represented by SEQ. ID. NO. 1 and NO. 2.