Dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection system and method for Rickettsia mooseri and Orientia tsutsugamushi

A technology for Rickettsia morii and Orientia tsutsugamushi, which is applied in the field of real-time fluorescence quantitative PCR detection methods and detection systems

Inactive Publication Date: 2015-11-04
宋锋林 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the process of sample detection, due to factors such as sample infection and operator technical level, traditional detection methods often have certain limitations.

Method used

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  • Dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection system and method for Rickettsia mooseri and Orientia tsutsugamushi
  • Dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection system and method for Rickettsia mooseri and Orientia tsutsugamushi
  • Dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection system and method for Rickettsia mooseri and Orientia tsutsugamushi

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Experimental program
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Embodiment 1

[0081] A dual real-time fluorescent quantitative PCR detection system for Rickettsia mosoni and Orientia tsutsugamushi, including primers and probes for Rickettsia mosoni and Orientia tsutsugamushi, wherein,

[0082] Rickettsia moschii primers and Taqman-MGB probe sequences:

[0083] Pr47F: 5'-TGTTGATGGTGCAGGATTTGA-3'

[0084] Pr47R: 5'-CGAATTTGTAGCGACAGGAAG A -3'

[0085] Probe-P: 5’-FAM-CAAACTGGCGCTGGTGT-3’

[0086] Orientia tsutsugamushi primers and Taqman-MGB probe sequences:

[0087] Ot-F: 5’-TCT RCR CCAGTAATYATTCCTCC-3’ R=A / G Y=T / C

[0088] Ot-R: 5'-TGTTAATTGCTAGTGCAATGTCTGC-3'

[0089] Probe-O: 5'-HEX-AAGGACCACACTCTAAT-3'.

[0090] The detection body system PCR reaction system:

[0091] TaqMan mix buffer 10 μL

[0092] Pr47F / R (10 μM) 0.4 μL

[0093] Ot-F / R (10 μM) 0.8 μL

[0094] Probe-P (10 μM) 0.6 μL

[0095] Probe-O (10 μM) 0.6 μL

[0096] pMD18-T-Rm 1 μL

[0097] pMD18-T-Ot 1 μL

[0098] complement ddH 2 0 to 20 μL;

[0099] Among them, the positive co...

Embodiment 2

[0104] Dual real-time fluorescent quantitative PCR detection method for Rickettsia mosoni and Orientia tsutsugamushi:

[0105] (1) First, the design and synthesis of primers and probes:

[0106] The upstream and downstream primers and Taqman-MGB probe were designed and synthesized based on the ompB protein gene sequence published by GenBank (GI: 51459527), and the amplified fragment size was 64bp.

[0107] Pr47F: 5'-TGTTGATGGTGCAGGATTTGA-3'

[0108] Pr47R: 5'-CGAATTTGTAGCGACAGGAAG A -3'

[0109] Probe-P: 5'-FAM-CAAACTGGCGCTGGTGT-3';

[0110] Based on the Karp-type gene sequence of Orientia tsutsugamushi published by GenBank (GI: 63079111), the 56-kDa protein gene sequence was designed and synthesized with upstream and downstream primers and Taqman-MGB probes. The amplified fragment size was 130bp.

[0111] Ot-F: 5’-TCT RCR CCAGTAATYATTCCTCC-3’ R=A / G Y=T / C

[0112] Ot-R: 5'-TGTTAATTGCTAGTGCAATGTCTGC-3'

[0113] Probe-O: 5'-HEX-AAGGACCACACTCTAAT-3';

[0114] (2) Sy...

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Abstract

The invention discloses a dual real-time fluorescence quantitative PCR (polymerase chain reaction) detection system and method for Rickettsia mooseri and Orientia tsutsugamushi. The detection system is composed of primers, probes, a TaqMan mix buffer, ddH2O, pMD18-T-Rm and pMD18-T-Ot, the primers include Pr47F, Pr47R, Ot-F and Ot-R, and the probes include a Probe-P and a Probe-O. The primers and the probes have the advantages of high specificity and high sensitivity. The detection method does not have cross reaction with Rickettsia prowazeki, Rickettsia rickettsii and Q fever rickettsia, and 8.24 copied Rickettsia mooseri standards and 43.6 copied Orientia tsutsugamushi standards can be detected at the same time. The dual real-time fluorescence quantitative PCR detection system and method can be used for detecting Rickettsia mooseri and Orientia tsutsugamushi at the same time and is suitable for frontier port health quarantine departments to conduct monitoring and detecting work of the above pathogens.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a real-time fluorescence quantitative PCR detection method and a detection system, which can simultaneously perform nucleic acid detection on Rickettsia moschii and Orientia tsutsugamushi. Background technique [0002] Arboborne diseases are an important class of infectious diseases, accounting for about 5% to 10% of the total incidence of infectious diseases in my country every year, and the number of deaths accounts for 30% to 40% of the total deaths of infectious diseases. At present, the prevalence and outbreak of arboviruses presents a new trend: the re-emergence of the original arboviruses and the continuous expansion of the endemic area; big. [0003] Murine typhus (Murine Typhus) is caused by Rickettsia mosoni ( Rickettsia mooseri ) is a natural foci disease transmitted by vector organisms such as rats and fleas; Tsutsugamushi Disease, also known as ju...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6851C12Q2531/113C12Q2561/101C12Q2545/113C12Q2561/113Y02A50/30
Inventor 高玉峰宋锋林万雪程晓兰姜陆
Owner 宋锋林
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