Nucleic acid sequence, kit and method used for isothermal amplification detection of yersinia pestis and application

A constant temperature amplification detection and Yersinia pestis technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of large investment costs and false positives, and achieve good applicability and steps Simple and improved detection sensitivity

Pending Publication Date: 2021-02-23
内蒙古自治区综合疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR technology has the advantages of high detection sensitivity, simple and fast operation, etc. However, its initial investment cost is relatively large (depending on the functional partition of the laboratory and the PCR instrument), and it is prone to false positives caused by laboratory contamination.

Method used

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  • Nucleic acid sequence, kit and method used for isothermal amplification detection of yersinia pestis and application
  • Nucleic acid sequence, kit and method used for isothermal amplification detection of yersinia pestis and application
  • Nucleic acid sequence, kit and method used for isothermal amplification detection of yersinia pestis and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The composition of the Yersinia pestis constant temperature amplification detection kit is as follows:

[0070] ②Isothermal amplification reaction solution: 20mM Tris-HCl (pH=8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 1MMgSO 4 , 10×Thermo pol buffer, 5M betaine, 8U / μl Bst DNA polymerase, 10mM dNTPs, 0.1μM each for SEQ ID No.1 and SEQ ID No.2, 0.3μM for SEQ ID No.3, and SEQ ID No.4 0.3 μM, SEQ ID No.5 0.6 μM; the primers and probes involved were synthesized at Shanghai Sangon Bioengineering Co., Ltd., and the sequences are shown in Table 1.

[0071] ③ Positive control template: a DNA fragment synthesized in vitro containing the pla gene of Yersinia pestis. The preparation method of the positive control template is as follows: perform PCR amplification on the genomic DNA of Yersinia pestis with downstream strand displacement primers and upstream strand displacement primers; purify the amplified products with Roche PCR product purification kit; measure A 260 Quantify and d...

Embodiment 2

[0074] The specific method of the kit to detect Yersinia pestis:

[0075] Step 2: Add the step DNA as a template into the PCR tube containing the constant temperature amplification reaction solution, add the positive control template and the negative control into the control PCR tube respectively, and take 2 μl of sample DNA and control DNA, and 18 μl of reaction solution , the total reaction volume is 20μl; the amplification reaction is carried out at 60°C for 90min;

[0076] Step 3: Put the reacted PCR tube into the nucleic acid test strip anti-pollution detection device for detection, and interpret the experimental results after 5 minutes: when the quality control line (C line) and the detection line (T line) of the test strip are both When it is red, it is positive, indicating that the sample contains Yersinia pestis nucleic acid; when the quality control line (C line) of the test strip is red, and the detection line (T line) is colorless, it is negative, indicating that t...

Embodiment 3

[0077] Embodiment 3 sensitivity evaluation

[0078] The in vitro synthetic DNA standard containing the Yersinia pestis pla gene fragment is diluted 10 times and then detected by the kit and detection method of the present invention.

[0079] The preparation method of the in vitro synthetic DNA standard substance containing Yersinia pestis pla gene fragment is:

[0080] The Pla gene fragment of Yersinia pestis was artificially synthesized, and a 456bp specific fragment was amplified by using primers SEQ ID NO: 1 to SEQ ID NO: 2. The A260 was quantified by a spectrophotometer, and then converted and diluted to 1.0×10 7 copy / mL. Store at -20°C as a working standard.

[0081] see results image 3 , where 1~8 are 10 respectively 7 ~10 0 Copy the DNA standard, 9 is the negative control.

[0082] Experimental results show that the detection sensitivity of the kit of the present invention to the nucleic acid of Yersinia pestis is 100 copies of DNA molecules / reaction, so as to me...

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Abstract

The invention relates to the field of pathogen microorganism detection, in particular to a nucleic acid sequence, kit and method used for isothermal amplification detection of yersinia pestis and application. The nucleic acid sequence of the invention includes strand displacement primer sequences as shown in SEQ ID No.1 and SEQ ID No.2, probes as shown in SEQ ID No.3 and SEQ ID No.4, and a cross amplification primer sequence as shown in SEQ ID No.5. The kit and the detection method of the invention have the technical advantages of high specificity, high sensitivity, simple steps and high repeatability.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular, to a nucleic acid sequence, kit, method and application for constant temperature amplification and detection of Yersinia pestis. Background technique [0002] Plague is a natural foci disease caused by the bacillus Yersinia pestis (Yersinia genus), which is widely prevalent in humans and rodents, and is mainly transmitted by ticks, fleas and other vectors. Currently, hundreds of species of animals in nature have been confirmed to be infected with plague. Due to factors in the ecological environment and diversification of modes of transmission, plague is widely prevalent in animals in nature, such as rodents, rabbits, and marmots. The spread of Yersinia pestis often spreads among rats first, leading to the death of a large number of rats. When the sick rats die, ticks turn their parasitic hosts to humans, and pass the plague to humans by sucking blood, thus causing human plag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107C12Q2565/625C12Q2563/131
Inventor 高雨龙范蒙光李建云温永俊郑利英
Owner 内蒙古自治区综合疾病预防控制中心
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