Loop-mediated isothermal amplification detection kit for rotavirus

A detection kit, ring-mediated isothermal technology, applied in the determination/inspection of microorganisms, methods based on microorganisms, microorganisms, etc., can solve the problems of low sensitivity, non-specificity, cumbersome operation, etc., and achieve high sensitivity and specificity strong effect

Inactive Publication Date: 2013-04-03
GUANGDONG INST OF MICROORGANISM +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although the prevalence of rotavirus gastroenteritis has a certain seasonality, it can occur throughout the year, and it can be an outbreak or a sporadic epidemic, so it cannot be effectively protected in a timely manner
[0004] At present, the detection methods of rotavirus pathogens include electron microscope technology, virus isolation and culture technology, immunology technology and molecular biology technology, but the first three technologies mentioned above often have low sensitivity, cumbersome and time-consuming operations, and require large and expensive instruments and equipment And other shortcomings, often h

Method used

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  • Loop-mediated isothermal amplification detection kit for rotavirus
  • Loop-mediated isothermal amplification detection kit for rotavirus

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: Detection of Rotavirus by Rotavirus LAMP Detection Kit

[0021] Preparation of samples to be tested: taking clinical diarrhea samples as an example

[0022] In this embodiment, 3 clinical diarrhea samples collected from hospitals were collected, and were tested according to the following methods.

[0023] (1) Sample processing

[0024] Take the clinical diarrhea samples collected in the hospital, dilute them into a 10% suspension with PBS at pH 7.4, centrifuge at 10,000g at 4°C to get the supernatant, and use it to extract viral RNA.

[0025] (2) Viral RNA extraction

[0026] (a) Take 200 μL of the supernatant from step (1), add 800 μL Trizol reagent, mix it several times with a pipette, and let stand for 5 minutes;

[0027] (b) Add 200 μL of chloroform, shake vigorously for 15 seconds, and then let stand for 2 to 3 minutes;

[0028] (c) 4°C, 12000g, centrifuge for 15min, discard the upper liquid;

[0029] (d) Add 500 μL of isopropanol, mix thoroughly, a...

Embodiment 2

[0041] Embodiment 2: specificity test

[0042] One known rotavirus-negative and two known rotavirus-positive samples were tested again according to the RT-LAMP method in Example 1, and the results showed that the known negative samples were still negative in the LAMP test results , the known positive samples are still positive in the LAMP detection result, indicating that the LAMP detection result is consistent with the PCR detection result, and the specificity of the present invention is verified.

Embodiment 3

[0043] Embodiment 3: sensitivity test

[0044]The rotavirus RNA was gradiently diluted, and reverse transcription, loop-mediated isothermal amplification, and color detection of the amplified product were performed according to the method of steps 3-5 in Example 1, to verify the sensitivity of the present invention, and the detection result was: negative The control (template is water) is yellow and does not contain rotavirus, the positive control (RT-PCR positive and confirmed positive by sequencing) is green and contains rotavirus, while 100pg, 10pg, 1pg, 0.1pg template Its color of the rotavirus RNA sample of amount is all green, is all positive, and it is determined that its detection sensitivity is 0.1pg.

[0045]

[0046]

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Abstract

The invention discloses a loop-mediated isothermal amplification detection kit for rotavirus. The detection kit comprises an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP, a downstream inner primer BIP and reaction liquids B and C, wherein the upstream outer primer F3, the downstream outer primer B3, the upstream inner primer FIP and the downstream inner primer BIP are expressed by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively. According to the invention, two specific inner primers and two specific outer primers are designed according to gene conserved sequences of the rotavirus; and the gene conserved sequences are shared by the rotavirus. The kit adopts the LAMP technology, so that the specificity is strong; the kit has higher sensitivity compared with the PCR detection method, but the expensive PCR amplifier is not needed, and all that is needed is the general water bath tank; and the result is not required to be observed by the gel electrophoresis method, but is observed by fluorescent dye, so that the operation is easy and fast; and the kit can be used for detecting the rotavirus, and is particularly suitable for basic level field application.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit for loop-mediated isothermal amplification of rotavirus. Background technique: [0002] Rotavirus is an important cause of viral gastroenteritis worldwide. At present, there is no specific drug for the treatment of gastroenteritis caused by rotavirus infection. No matter in developed or developing countries, diarrhea caused by rotavirus has a high incidence rate and is the most important cause of morbidity and death in infants and young children. [0003] Rotavirus has strong viability in vitro, strong resistance to various physical and chemical factors, resistance to ether and weak acid, and cannot be inactivated by chloroform, repeated freezing and thawing, and ultrasonic treatment. The sequence variation is large, and there are many serotypes. In recent years, the prevalence of gastroenteritis caused by rotavirus has been on the rise worldwide, with...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 寇晓霞吴清平薛亮张菊梅
Owner GUANGDONG INST OF MICROORGANISM
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