Anaplasma and Rickettsia real-time fluorescent quantitative PCR kit and method

A real-time fluorescence quantitative and fluorescence quantitative technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, and microbial determination/examination, etc., can solve the problem of insufficient understanding of laboratory diagnostic technology, multiple organ involvement and death of patients, and easy misdiagnosis and missed diagnosis. and other problems, to achieve the effect of high sensitivity, good specificity, and easy operation.

Inactive Publication Date: 2011-12-21
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Due to the lack of understanding of this type of disease and the lack of necessary laboratory diagnostic techniques, clinical misdiagnosis and missed diagnosis are easy to occur, often leading to the death of patients with multiple organ involvement
At present, the clinical diagnosis of such diseases in my country still mainly relies on clinical manifestations and empirical treatment, and a few better units still use traditional Wai-Field's reaction with poor specificity.

Method used

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  • Anaplasma and Rickettsia real-time fluorescent quantitative PCR kit and method
  • Anaplasma and Rickettsia real-time fluorescent quantitative PCR kit and method
  • Anaplasma and Rickettsia real-time fluorescent quantitative PCR kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: detect the real-time fluorescent quantitative PCR kit of Anaplasma

[0063] 1. Primer design and probe synthesis

[0064] According to the Anaplasma msp2 gene sequence (EF143798) provided by GenBank, BLAST comparison was performed to design probes and primers. The designed probes can detect 39 different isolates. Probes and primers (synthesized by Shanghai Jikang Company) were designed using ABI primer Express 2.0 software. See Table 1 for specific primers, probe names and sequences, and GeneBanK acceptance numbers of detectable strains.

[0065] Table 1 Primers, probes and detectable strains used in detection of Anaplasma msp2 gene

[0066]

[0067]

[0068] 2. Standard DNA template preparation and sensitivity analysis

[0069] The MPF and MPR primer pairs in Table 1 were used to amplify 159bp fragments using the A. phagocytophilum Webster strain as a template, and the PCR products were purified (QIAgen, German) and connected to the PGM-T cloning...

Embodiment 2

[0074] Example 2: Real-time fluorescent quantitative PCR kit for detecting Orientia tsutsugamushi

[0075] 1. Primer design and probe synthesis

[0076] According to the conserved sequences of dominant 56KD specific genes such as Karp (M33004.1), Kato (GenBank) and Gillim (DQ789360.1), which are common in Orientia tsutsugamushi in my country provided by GenBank, BLAST comparison was performed to design probes and primers, and design probes and primers. Probes and primers were designed using ABI primer Express 2.0 software.

[0077] Table 2 Primers, probes and detectable strains used in the detection of Orientia tsutsugamushi 56KD specific genes

[0078]

[0079] 2. Standard DNA template preparation and sensitivity analysis

[0080] The WF and WR primer pairs in Table 2 were used to amplify a 361bp fragment using the Orientia tsutsugamushi Karp strain as a template, and the PCR product was purified (QIAgen, German) and connected to the PGM-T cloning vector and transformed ...

Embodiment 3

[0085] Embodiment 3: detect the real-time fluorescent quantitative PCR kit of spotted fever group Rickettsia

[0086] 1. Primer design and probe synthesis

[0087] By comparing the sequences of 14 species of spotted fever group Rickettsia SFGR ompA on GenBank, ABI primer Express 2.0 software was used to design probes and primers for fluorescent quantitative PCR (synthesized by Shanghai Jikang Company). The specific primers, probes and construction standard plasmid primer sequences of fluorescent quantitative PCR are shown in Table 3.

[0088] Table 3 Fluorescent quantitative PCR-specific primers and construction standard plasmid primers for spotted fever group Rickettsia ompA gene

[0089]

[0090] 2. Standard DNA template preparation and sensitivity analysis

[0091] The primers Rh-F and Rh-Rr amplified a 305bp fragment from the R.heilongjiangii-054 strain DNA (the strain is a native isolate in my country, which was isolated from the forest tick in Heilongjiang Province ...

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Abstract

The invention discloses a real-time fluorescence quantitative polymerase chain reaction (PCR) kit for five kinds of pathogens such as incorporeity, rickettsia and the like and a method thereof. Specific oligonucleotide primers and probes are designed according to the specific gene nucleotide sequences of the five kinds of pathogens such as the incorporeity, the rickettsia and the like. The invention provides a method for detecting the incorporeity and the rickettsia and a high-sensitivity detection kit consisting of the specific primers. The kit is easy, convenient and quick in operation, andhas high sensitivity and specificity. The kit can perform rapid specific detection on 5 kinds of related rickettsia and is applicable to clinical laboratories and disease monitoring tissue laboratories. The invention also discloses a method for detecting the incorporeity and the rickettsia by using the kit.

Description

technical field [0001] The invention relates to a detection kit and a method for five kinds of pathogens such as anaplasma and rickettsia. Specifically, the present invention relates to the design of specific oligonucleotide primers and probes based on the highly conserved nucleotide sequences of specific genes of five pathogens such as Anaplasma and Rickettsia, which are assembled by real-time fluorescent quantitative PCR technology. The detection kit for Anaplasma and Rickettsia, and the invention also relates to the detection method thereof. Background technique [0002] Anaplasma, Orientia tsutsugamushi, Rickettsia spotted fever group, typhus, and Coxiella beinii are all pathogenic members of Rickettsiae. [0003] Anaplasmosis is a new tick-borne natural foci rickettsial disease that seriously threatens the health of humans and animals. It is caused by the infection of Anaplasma phagocytophilum. At present, the US CDC has listed the disease as a legally notifiable infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 张丽娟亚红祥梁长威
Owner ICDC CHINA CDC
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