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Fluorescent PCR reagent and method for detecting yersinia pestis

A Yersinia pestis and fluorescence technology, applied in the field of nucleic acid detection of Yersinia pestis, can solve problems such as missed detection of Yersinia pestis, and achieve the effect of ensuring accuracy, avoiding failure, and good inclusiveness

Inactive Publication Date: 2021-04-06
深圳市赛格诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This means that there is a certain risk of missed detection in the detection of Yersinia pestis by targeting the pla gene and caf1 gene

Method used

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  • Fluorescent PCR reagent and method for detecting yersinia pestis
  • Fluorescent PCR reagent and method for detecting yersinia pestis
  • Fluorescent PCR reagent and method for detecting yersinia pestis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: This example provides a design scheme of fluorescent PCR primers and probes for detecting Yersinia pestis nucleic acid.

[0037] The present invention selects by document research and GenBank nucleic acid database analysis figure 1 The dam gene nucleic acid sequence shown and figure 2The nucleic acid sequence of the pla gene shown is also used as the detection target sequence of Yersinia pestis. The pPCP1 plasmid where the pla gene is located has a high copy number in Yersinia pestis cells, so it has high detection sensitivity for most Yersinia pestis strains; considering that some Yersinia pestis strains do not contain pPCP1 plasmids, At the same time, using the dam gene on the chromosome as the detection target can prevent missed detection due to missing plasmids.

[0038] Use the software Primer Express V3.0 to design the following primers for amplifying the target sequence and the probe sequences for detecting the target sequence:

[0039] The sequen...

Embodiment 2

[0043] Example 2: This example demonstrates the preparation of fluorescent PCR reagents for Yersinia pestis.

[0044] Yersinia pestis fluorescent PCR reagent provided by the present invention is designed and prepared by 25 μ l reaction volume, and the components are shown in Table 1, and each component (potassium chloride, magnesium chloride, dNTPs, Taq DNA polymerase, primer and probe) is carried out gradient dosage Optimize, finally determine the dosage shown in Table 1, and then prepare 2x (ie 12.5 μl) fluorescent PCR liquid detection reagent for Yersinia pestis according to the ingredients and content described in Table 1.

[0045] On the basis of liquid reagents, add freeze-dried excipients (Table 1), divide them into PCR eight-tube tubes according to the amount of one reaction per tube, and then freeze-dry to obtain the freeze-dried fluorescent PCR of Yersinia pestis. The detection reagent is a white particle ( image 3 ). Add 23 μl of nuclease-free water to a portion ...

Embodiment 3

[0046] Example 3: This example demonstrates the detection effect of Yersinia pestis nucleic acid dam gene and pla gene positive reference products using the Yersinia pestis freeze-dried fluorescent PCR detection reagent of the present invention.

[0047] After mixing equal amounts of Yersinia pestis dam gene and pla gene positive reference materials, carry out 10-fold serial dilution, take 23 μl of each concentration and add to 1 part of Yersinia pestis lyophilized fluorescent PCR detection prepared according to Example 2 reagent; 23 μl of nuclease-free water was used as a negative control. The resulting 25 μl reaction mixture was dissolved by slow shaking, centrifuged, and detected on a fluorescent PCR instrument. The reaction program was set according to Table 2. Each treatment was repeated 3 times.

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Abstract

The invention provides a fluorescent PCR reagent and method for detecting yersinia pestis. The yersinia pestis fluorescent PCR detection reagent provided by the invention takes a dam gene on a yersinia pestis chromosome and a pla gene on a pPCP1 plasmid as detection targets, has relatively high detection sensitivity on most strains, and ensures that individual strains are not missed due to deletion of the pla gene. Primer and probe sequences used by the reagent are designed and screened on the basis of latest yersinia pestis nucleic acid sequence information, and have good inclusivity, specificity and reaction performance, and the detection accuracy can be ensured. Preferably, the yersinia pestis fluorescent PCR detection reagent is prepared into a freeze-dried reagent by using a freeze-drying technology, and the freeze-dried reagent is stable at normal temperature and does not need cold-chain transportation and storage, so that the risk of failure of a conventional liquid fluorescent PCR detection reagent due to storage temperature change or repeated freeze thawing is avoided; and the kit meets the use requirements of regions with long-distance transportation and poor cold chain conditions, and can be used for on-machine detection by directly adding a nucleic acid sample during use.

Description

technical field [0001] The invention relates to pathogenic microorganism detection technology, in particular to nucleic acid detection of Yersinia pestis. Background technique [0002] Plague is a Class A infectious disease stipulated in the Law on the Prevention and Control of Infectious Diseases in my country, and its pathogen is Yersinia pestis. Plague can be divided into bubonic plague, septicemic plague, pneumonic plague, intestinal plague, meningitis-type plague, ocular plague, cutaneous plague, etc. according to the location of the disease, among which bubonic plague is the most common. The main transmission routes of plague are: (1) flea bites. The main source of infection for plague is rodents, and Yersinia pestis can be transmitted to humans and other animals through flea bites. The first cases of human plague were mostly caused by the bite of Xenopsylla cheopis, a parasitic house mouse. Yersinia pestis enters the human body through the skin and spreads along th...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2563/107
Inventor 黎绍基林镜中黄俊辉朱碧莹刘钰涵
Owner 深圳市赛格诺生物科技有限公司
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