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A kind of rickettsia multiplex fluorescent PCR detection method

A technology of multiple fluorescence and detection methods, applied in the field of PCR detection, can solve the problems of poor sensitivity, stability and specificity of multiple PCR, and achieve the effect of improving sensitivity and shortening the detection cycle

Inactive Publication Date: 2019-04-16
四川国际旅行卫生保健中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The invention provides a multiplex fluorescent PCR detection method for Rickettsia, which intends to combine the multiplex PCR method with the fluorescent quantitative method to solve the problems of poor sensitivity, stability and specificity of multiplex PCR detection, and overcome the cost of single-fold fluorescent PCR. Due to the disadvantages of time and time, PCR detection can not only diagnose a variety of common Rickettsia pathogens at the same time, but also meet the needs of epidemiological investigation and rapid detection

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  • A kind of rickettsia multiplex fluorescent PCR detection method
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  • A kind of rickettsia multiplex fluorescent PCR detection method

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Embodiment 1

[0061] A kind of rickettsia multiplex fluorescent PCR detection method provided by the invention comprises the following steps:

[0062] (1) Extract DNA, using the classic phenol-chloroform method to extract DNA, the specific steps are as follows:

[0063] ①Put an appropriate amount of animal tissue into a 0.5 mL centrifuge tube, place in 200 µL TE buffer, vortex, centrifuge, and discard the supernatant; then add 200 µL TE and soak for 1 to 3 h to remove the influence of ethanol (fresh samples do not Soaking is required; specimens more than 2 years old are soaked overnight), centrifuged, and the supernatant discarded;

[0064] ② Add 10 µL of STE+PK solution, grind it to a solution state with a melted pipette tip, then add 190 µL (390 µL if the sample size is large) of STE+PK solution, mix well to separate the tissue; place the centrifuge tube at 55 Digest in a water bath on a rotary mixer for 2 to 3 hours to digest protein;

[0065] ③ Add an equal volume of saturated phenol,...

Embodiment 3

[0150] (7) Example 3 Methodological Verification of Multiplex Fluorescent PCR Reaction System

[0151] ①Sensitivity verification, dilute about 10copies / mL plasmid stock solution into 10 5 、10 4 , 5×10 3 、10 3 , 5×10 2 Concentration, the optimized system determines the sensitivity of the detection method, the diluted positive plasmid is used as the detection sample, and each concentration sample is repeated twice for each group;

[0152] Depend on Figure 15 , Figure 16 and Figure 17 It can be seen that the sample concentration is 5×10 3 When copies / mL, the positive detection rate is 100%; when the sample concentration is 1×10 3 When copies / mL, some samples were not detected; when the sample concentration was 5×10 2 When copies / mL, the sample positive rate was 0%, as shown in Table 12.

[0153] Table 12 Summary table of sensitivity verification results of three kinds of Rickettsia

[0154]

[0155] Note: N / A means not detected.

[0156] Take the Ct value as the...

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Abstract

The invention discloses a multiplex fluorescence PCR (polymerase chain reaction) detection method for rickettsia, and relates to the field of PCR detection. A multiplex real-time fluorescence quantitative PCR method for three kinds of rickettsia of epidemic typhus, endemic typhus and Q fever is established by exploratory research on multiplex fluorescence PCR in multiple aspects of specificity of a primer probe, a key factor of a reaction system, quality control construction and the like, the detection sensitivity and specificity of the three kinds of rickettsia are obviously improved, and meanwhile, detection cycles for pathogens of the three kinds of rickettsia are greatly shortened; the method is significant for increasing the quarantine clearance speed of frontier ports, preventing infectious diseases from being brought in and out at the frontier ports, successfully coping with sudden public health events and protecting the life and property safety of people.

Description

technical field [0001] The invention relates to the field of PCR detection, in particular to a method for multiple fluorescent PCR detection of rickettsiae. Background technique [0002] Rickettsia is a prokaryotic microorganism, the cells are mostly club-shaped, the size is between bacteria and viruses, and it is obligate to parasitize in eukaryotic cells (except for a few). In nature, it mainly breeds in storage hosts such as rodents (mice) and livestock (cattle, sheep, dogs). Lice, fleas, ticks, mites and other blood-sucking arthropods are the main vectors. [0003] Rickettsial disease caused by Rickettsia is a zoonotic natural foci infectious disease that seriously threatens human health. The main clinical manifestations of rickettsial disease are non-specific clinical symptoms such as fever, headache, general malaise and rash (except Q fever), which are acute. Due to the lack of typical clinical manifestations, it is easy to be misdiagnosed as viral diseases and feve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12Q1/689C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2545/113C12Q2561/101Y02A50/30
Inventor 赵锋刘杨倪蓉王俊贤田绿波王宁
Owner 四川国际旅行卫生保健中心
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