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

A technology of Orientia tsutsugamushi and primer pairs, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low sensitivity, expensive testing equipment, long testing time, etc. Diagnostic accuracy, improving diagnostic accuracy, overcoming the effect of expensive equipment

Active Publication Date: 2015-01-14
金东民
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the above methods still have the following disadvantages: false positives caused by cross-reactions with other pathogenic microorganisms; it takes several days from the onset of disease symptoms until antibodies are formed, resulting in low sensitivity of early detection of the disease; and the need for additional follow-up tests to confirm the diagnosis
Because when conventional PCR is performed using 47kDa or 56kDa genes as targets, the sensitivity is as low as at most 10%
However, nested PCR has the disadvantage that it does require a longer test time than other PCRs since the PCR is performed twice to increase sensitivity
Meanwhile, real-time PCR has the disadvantage that it is limited in general use in most medical institutions due to the need for very expensive testing equipment

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Preparation of primers

[0037] The primer pair was designed based on the base sequence of the 16s ribosomal RNA gene amplified from the blood samples of patients infected with Boryoung strain (this strain is very common in Korea). However, it can also be used for other model strains (such as Gilliam, Karp, Kato) and various other strains. More specifically, the primer pairs were prepared as forward primers (positions 403-428) and reverse primers (positions 577-601) for GenBank accession number bankit1356164HM352765: Orientia tsutsugamushi Boryoung genotype SEQ.ID.NO.1 and SEQ.ID.NO.2 represent. Forward primers and reverse primers for other regions were also prepared, and the primers were represented by SEQ.ID.NO.3-SEQ.ID.NO.36 (see Table 1).

[0038] [Table 1]

[0039]

Embodiment 2

[0040] Example 2: Sample preparation and vital statistics

[0041] From 2007 to 2008, among patients 18 years of age or older who came to the hospital due to acute fever within 4 weeks of the onset, patients who exhibited at least two symptoms selected from the group consisting of the following symptoms were selected Come to take a blood sample: eschar, erythematous papules, headache, general collapse, muscle pain, cough, nausea, and stomach pain.

[0042] For blood samples in which the IFA titer of Orientia tsutsugamushi was increased by at least four times, patients with these samples were diagnosed as tsutsugamushi. Therefore, such patients are classified into the tsutsugamushi group (tsutsugamushi group). In patients in the fever control group (non-tsutsugamushi group), the indirect fluorescent antibody against Orientia tsutsugamushi was not detected in their blood, but they passed tests (such as serological tests, culture tests, and peripheral blood smear tests, etc.) ), the...

Embodiment 3

[0048] Example 3: PCR diagnosis using the primer pair of the present invention

[0049] In order to confirm the effectiveness of the prepared primer pair and the corresponding inspection method, DNA was extracted from the blood sample of the tsutsugamushi patient prepared in Example 2. In particular, whole blood is taken from the patient. Separate the buffy coat. Then, DNA was isolated by using QIA amp DNA mini kit (Qiagen, Germany). Separate by following the protocol attached in the manual. The isolated DNA was used as a template for detecting Orientia tsutsugamushi.

[0050] As a method for PCR diagnosis, conventional PCR is selected in this article, and the extracted DNA is used as a template. For the PCR, a 20μl reaction mixture was prepared by adding the following ingredients to AccuPowerTM PCR PreMix (1U Top polymerase, 250μM dNTP, 10mM Tris-HCl (pH9.0); Bioneer, Daejeon, Korea): 2μl template DNA, 1μl5pmol / μl forward primer and 1 μl 5pmol / μl reverse primer, and 16 μl st...

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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

Technical field [0001] The present invention relates to a primer pair for diagnosing Orientia tsutsugamushi (Orientia tsutsugamushi) and a method for diagnosing Orientia tsutsugamushi using the primer pair. More specifically, the present invention relates to a method for diagnosing Orientia tsutsugamushi (orientia tsutsugamushi). (Scrub typhus)) A primer pair that amplifies the base sequence of the 16s ribosomal RNA gene and a method for detecting tsutsugamushi disease by detecting Orientia tsutsugamushi using the primer pair. Background technique [0002] Tsutsugamushi is an acute febrile disease, which is transmitted by chiggers infected with Orientia tsutsugamushi and shows characteristic clinical symptoms due to systemic vasculitis. Rodents are the main host, and juvenile mites (chiggers) are the host and vector. [0003] Since tsutsugamushi disease was first reported in Korea in 1951, this major autumn fever has spread throughout the country, especially between September and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6883C12Q1/689C12Q1/6844
Inventor 金东民
Owner 金东民
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