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

A technology of Orientia tsutsugamushi and primer pairs, which is applied to biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., can solve the problems of expensive testing equipment, low sensitivity, false positives, etc., and improve diagnosis Accuracy, Excellent Diagnostic Accuracy, Effect of High Diagnostic Accuracy

Active Publication Date: 2013-05-01
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: Primer Preparation

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

[0038] [Table 1]

[0039]

Embodiment 2

[0040] Example 2: Sample Preparation and Vital Statistics

[0041] From 2007-2008, among patients aged 18 or older who came to the hospital due to acute febrile illness within 4 weeks from the onset of onset, patients exhibiting at least two symptoms selected from the group consisting of the following symptoms were selected Come for a blood sample: eschar, erythematous papules, headache, generalized prostration, muscle pain, cough, nausea, and stomach pain.

[0042] For blood samples whose IFA titers of Orientia tsutsugamushi increased at least four-fold, patients with these samples were diagnosed with tsutsugamushi. Therefore, such patients were classified into the scrub typhus patient group (the scrub typhus group). In patients in the febrile control group (non-tsutsugamushi group), indirect fluorescent antibodies against Orientia tsutsugamushi were not detected in their blood, while passing tests (such as serological tests, culture tests, and peripheral blood smear tests, ...

Embodiment 3

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

[0049] In order to confirm the effectiveness of the prepared primer pairs and corresponding inspection methods, DNA was extracted from the blood samples of scrub typhus patients prepared in Example 2. In particular, whole blood is taken from the patient. The buffy coat was separated. Then, DNA was isolated by using the QIA amp DNA mini kit (Qiagen, Germany). Isolation was performed by following the protocol attached in the manual. The isolated DNA was used as a template for the detection of Orientia tsutsugamushi.

[0050] As a method for PCR diagnosis, conventional PCR was selected here, and the extracted DNA was used as a template. For the PCR, a 20 μl reaction mixture was prepared by adding the following components to AccuPower™ PCR PreMix (1 U Top polymerase, 250 μM dNTPs, 10 mM Tris-HCl (pH 9.0); Bioneer, Daejeon, Korea): 2 μl template DNA, 1 μl 5 pmol / μl of forward primer and 1 μl of 5 pmol / μl re...

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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, and more specifically, the present invention relates to a method capable of treating Orientia tsutsugamushi (causing A primer pair for amplifying the base sequence of the 16s ribosomal RNA gene (the cause of scrub typhus) and a method for detecting scrub typhus by using the primer pair to detect Orientia tsutsugamushi. Background technique [0002] Scrub typhus is an acute febrile disease that is transmitted by chiggers infected with Orientia tsutsugamushi and shows characteristic clinical symptoms due to systemic vasculitis. Rodents are the main host, and larvae (chiggers) are the host and medium. [0003] Since scrub typhus was first reported in Korea in 1951, this major fall fever has spread across the country, especially during the September-November period. In part...

Claims

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Application Information

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