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Method for detecting Yersinia pestis by utilizing a loop-mediated isothermal amplification (LAMP)

A technology of Yersinia pestis and a new method, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problem of low sensitivity and specificity, time-consuming, unsuitable epidemic focus monitoring and On-site preliminary screening and other issues

Inactive Publication Date: 2010-03-10
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological methods can only be used for retrospective diagnosis. Although the four-step test is reliable, it takes too long
Conventional detection methods are easy to detect typical Yersinia pestis, but it is difficult to detect atypical Yersinia pestis, and the sensitivity and specificity are not high
PCR technology mostly uses the virulence genes existing on the Yersinia pestis plasmid as the target site for detection, and the investigation of atypical Yersinia pestis lacks specificity, and most PCR amplification methods are limited to indoors and are not suitable for the epidemic focus. Monitoring and on-site screening

Method used

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  • Method for detecting Yersinia pestis by utilizing a loop-mediated isothermal amplification (LAMP)
  • Method for detecting Yersinia pestis by utilizing a loop-mediated isothermal amplification (LAMP)
  • Method for detecting Yersinia pestis by utilizing a loop-mediated isothermal amplification (LAMP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Optimizing experiment of ratio condition of YP0392 inner and outer primer pairs

[0056] Most of the products of LAMP are amplified by FIP and BIP. The inner primer plays an important role in guiding the reaction process of DNA, while the outer primer mainly plays a role in the formation of circular structure DNA. The inner primer has a greater impact on the reaction. For the amount of external primers (0.2uM), select external primers: internal primers in the range of 1:2-1:12 to optimize the conditions; the specific internal and external primer ratio condition optimization reaction system is shown in Table 2, and the ratio of internal and external primers is shown in Table 3.

[0057] Table 2 YP0392 internal and external primer ratio condition optimization reaction system

[0058]

[0059] Table 3. YP0392 internal and external primer ratio conditions optimize the amount of internal primers

[0060]

[0061] Result: YP0392 primers can be amplifi...

Embodiment 2

[0062] Example 2: Optimizing experiment of ratio condition of YP2088 inner and outer primer pairs

[0063] Use the optimized value of the enzyme concentration condition to carry out the YP2088 internal and external primer ratio condition experiment.

[0064] Table 4. Optimal reaction system of YP2088 internal and external primer ratio conditions

[0065]

[0066] Results: The YP2088 primer pair began to appear amplified bands at 1:6, preferably the ratio of outer primers: inner primers was in the range of 1:6-1:10, more preferably 1:7-1:9, most preferably 1:8, 1:8.5, 1:8.8.

Embodiment 3

[0067] Embodiment 3: Mg2+ condition optimization experiment of YP0392

[0068] The 1×Thermol buffer delivered by Bst DNA polymerase contains 2mM Mg2+. In order to ensure the effectiveness of the reaction and enhance the sensitivity of the experiment, the Mg2+ concentration is selected to be 2mM-10mM (blank B, 2mM, 4Mm, 5mM, 6mM, 8mM, 10mM) Carry out the single factor condition experiment, the reaction operation 1.2.7.1, the reaction system is prepared and divided into packages, each tube is 15ul, and the label is marked. The amount of the reaction system is shown in Table 5, and the amount of Mg2+ added is shown in Table 6.

[0069] Table 5. YP0392Mg 2+ Condition experiment reaction system dosage

[0070]

[0071] Table 6. Mg 2+ Conditional Experiment Response Mg 2+ scale

[0072]

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Abstract

The invention relates to a novel method for detecting Yersinia pestis, the loop-mediated isothermal amplification LAMP is adopted. The invention substantially improves a loop-mediated isothermal amplification method to enhance the detecting specificity and sensitivity.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to an improved loop-mediated isothermal amplification method and its application in detecting Yersinia pestis. Background technique [0002] Plague is a natural foci infectious disease characterized by acute onset, rapid spread, high fatality rate, and strong infectivity. Since the 1990s, the global plague epidemic has been on the rise. Plague epidemics have continued throughout the world year after year, especially in Africa, followed by Asia, but there are also large-scale outbreaks. Like other parts of the world, the natural foci of plague in my country have entered an active period, and the plague epidemic has also increased significantly. The pathogen of plague, Yersinia pestis, often presents the coexistence of typical bacteria and atypical bacteria in nature, and atypical bacteria is a strain that does not have one or several specific biological characteristi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
Inventor 王静文海燕龙冬伶孙肖红杨宇张晓龙姚李四
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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