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Primer, detection method and detection reagent kit for detection of group A type G3 rotavirus

A technology of rotavirus and detection method, which is applied in the direction of biochemical equipment and method, microorganism measurement/inspection, etc., can solve the problems such as the detection method and detection kit that have not detected the G3 type of rotavirus group A, and achieves Wide application range, strong specificity and high sensitivity

Inactive Publication Date: 2008-04-02
ZHUHAI DISEASE PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on detection methods and kits for detection of rotavirus group A G3 type by using loop-mediated isothermal amplification method, as well as LAMP primers and primer sets for specific gene fragments of rotavirus group A G3 type

Method used

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  • Primer, detection method and detection reagent kit for detection of group A type G3 rotavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Amplification of the known rotavirus group A G3 VP7 gene

[0047] 1) Design of primer set

[0048]The 307---505bp nucleic acid sequence of the rotavirus group A G3 type specific gene VP7 was screened out by consulting the literature and using BLAST software analysis, and six sites (these six sites were respectively: 307-324bp, 307-324bp, 332-356bp, 372-393bp, 417-440bp, 463-485bp, 488-505bp) LAMP primers were designed and synthesized to obtain the following primers; primer design was completed by LAMP special primer design software combined with molecular biology analysis software Advance Vector NTI .

[0049] serial number 1

[0050] Forward Outer Primer F3-G3 ACTGAAGCAGCAACAGAA

[0051] serial number 2

[0052] Reverse outer primer B3-G3 ACATGTCCAGTTGCAGTG

[0053] serial number 3

[0054] Forward inner primer FIP-G3

[0055] AGATCCTGTTGGCCATCCTTTAATAATTCATGGAAGGATACACTTTC

[0056] serial number 4

[0057] reverse inner primer BIP-G3

[0058] TATTG...

Embodiment 2

[0096] The difference between this example and Example 1 is that in this example, the reaction system used for gene amplification based on the LAMP method is:

[0097] The reaction system is: (the total reaction volume is 25ul)

[0098] Element

stock solution concentration

Quantity (ul)

Final concentration

nucleic acid template

FIP-G3

BIP-G3

F3-G3

B3-G3

betaine

dNTP

MgSO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

AMV reverse transcriptase

wxya 2 o

25uM

25uM

7.5uM

7.5uM

4M

10mM

100mM

10×

8U / ul

20U / ul

1.0

1.0

1.0

0.5

0.5

5.0

2.5

0.5

2.5

0.5

0.5

9.5

1.0uM

1.0uM

0.15uM

0.15uM

0.8M

1.0mM

2.0mM

0.16U / ul

0.4U / ul

[0099] In addition to the nucleic acid template, the above reaction system c...

Embodiment 3

[0106] The difference between this example and Example 1 is that in this example, the reaction system used for gene amplification based on the LAMP method is:

[0107] The reaction system is: (the total reaction volume is 25ul)

[0108] Element

stock solution concentration

Quantity (ul)

Final concentration

nucleic acid template

FIP-G3

BIP-G3

F3-G3

B3-G3

betaine

dNTP

MgSO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

AMV reverse transcriptase

wxya 2 o

50uM

50uM

15uM

15uM

7.5M

10mM

150mM

10×

16U / ul

20U / ul

1.0

1.0

1.0

0.5

0.5

5.0

4.0

1.0

2.5

1.0

0.5

7.0

2.0uM

2.0uM

0.3uM

0.3uM

1.5M

1.6mM

6.0mM

0.64U / ul

0.4U / ul

[0109] In addition to the nucleic acid template, the above reaction system can ...

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Abstract

The present invention relates to a fast analytical technique on foodborn pathogens based on loop-mediated isothermal amplification (LAMP), and provides a primer (or primers) which is (are) used for analysis on G3 of group A of rotavirus, and capable of amplifying the specific base sequence of the target gene, which is the VP7 gene (accession number of GenBank: D86272) of G3 of group A of rotavirus, and the primer(s) is (are) complemented with a partial sequence on the sites from 5095 to 5319 of the target gene, or with the complementary chain of the partial sequence. The present invention provides a group of primers or a primer with specificity on the specific gene segments of G3 of group A of rotavirus, with the utilization of the kit comprising the group of primers to analyze whether a specific gene segment of G3 of group A of rotavirus exists in the sample under analysis, thereby identifying whether G3 of group A of rotavirus exists in the sample.

Description

technical field [0001] The invention relates to a rapid detection technology for food-borne pathogens based on a loop-mediated isothermal amplification (LAMP) technology. In particular, it relates to a primer and a primer set specific to a specific gene fragment of the rotavirus group A G3 type; it also relates to a detection method for detecting the rotavirus group A G3 type by using the primer and the primer set with a loop-mediated isothermal amplification method and test kits. Background technique [0002] High incidence of foodborne illness, caused by Salmonella, Shigella, Staphylococcus aureus, Proteus, Vibrio cholerae, Vibrio parahaemolyticus and E.coli O157:H7, rotavirus and norovirus Food poisoning caused by rotavirus accounts for a very high proportion of the incidence of foodborne diseases in my country and is a serious public health problem. Most food poisoning caused by rotavirus is caused by G3 type. [0003] At present, the detection of foodborne pathogens ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 魏泉德张彩虹谭爱军张丽荣
Owner ZHUHAI DISEASE PREVENTION & CONTROL CENT
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