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Method for detecting DNA of schistosoma through fluorescent probe

A fluorescent probe, schistosomiasis technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as false positives, long time consumption, contamination of amplification products, etc., and achieve simple operation, easy collection, and short time. Effect

Active Publication Date: 2017-05-24
JIANGSU INST OF PARASITIC DISEASES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR and Q-PCR methods rely on thermal cycle instruments, have high requirements on the operating environment and personnel, and take a long time. The problem with the LAMP method is that the amplification products will pollute the environment, which will lead to false positives and affect the operation. The requirements for the environment and operating skills are higher, and these methods are not suitable for application in grassroots units and on-site inspections

Method used

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  • Method for detecting DNA of schistosoma through fluorescent probe
  • Method for detecting DNA of schistosoma through fluorescent probe
  • Method for detecting DNA of schistosoma through fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Sample source: Genomic DNA extracted from adults of Schistosoma japonicum, the concentration of DNA is 50ng / μL.

[0071] Design the following schistosome primers and probes, and entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize them:

[0072] Forward primer sequence: 5'-TACCTCAAGAAGTAATGTCCTTCCATTGTG-3',

[0073] Reverse primer sequence: 5'-ATGCGAGGTTTCAGGAGACCAAGAAGAACG-3'.

[0074] The sequence of the fluorescent probe is:

[0075] 5'CATAGGAGGTCATCTTGTTCAAGGTCAAGTCTCACCATCAACTCTTA-3'

[0076] Prepare the amplification system

[0077] Prepare the isothermal nucleic acid amplification system (volume 50 μL) in a 200 μL centrifuge tube according to the following ratio:

[0078]

[0079]

[0080] The above-prepared amplification system was lyophilized under negative pressure in a freeze dryer to become a powdery amplification system.

[0081] Add polyethylene glycol with a final concentration of 6% (w / v) and a molecular weight of 35,000 to the c...

Embodiment 2

[0083] Sample source: Genomic DNA extracted from adults of Schistosoma japonicum, the concentration of DNA is 70ng / μL.

[0084] Design the following schistosome primers and probes, and entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize them:

[0085] Forward primer sequence: 5'-TACCTCAAGAAGTAATGTCCTTCCATTGTG-3',

[0086] Reverse primer sequence: 5'-ATGCGAGGTTTCAGGAGACCAAGAAGAACG-3'.

[0087] The sequence of the fluorescent probe is:

[0088] 5'CATAGGAGGTCATCTTGTTCAAGGTCAAGTCTCACCATCAACTCTTA-3'

[0089] Prepare the amplification system

[0090] Prepare the isothermal nucleic acid amplification system (volume 100 μL) in a 200 μL centrifuge tube according to the following ratio:

[0091]

[0092]

[0093] The above-prepared amplification system was lyophilized under negative pressure in a freeze dryer to become a powdery amplification system.

[0094] Add polyethylene glycol with a final concentration of 6% (w / v) and a molecular weight of 35,000 to the ...

Embodiment 3

[0096] Sample source: Genomic DNA extracted from adults of Schistosoma japonicum, the concentration of DNA is 82ng / μL.

[0097] Design the following schistosome primers and probes, and entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize them:

[0098] Forward primer sequence: 5'-TACCTCAAGAAGTAATGTCCTTCCATTGTG-3',

[0099] Reverse primer sequence: 5'-ATGCGAGGTTTCAGGAGACCAAGAAGAACG-3'.

[0100] The sequence of the fluorescent probe is:

[0101] 5'CATAGGAGGTCATCTTGTTCAAGGTCAAGTCTCACCATCAACTCTTA-3'

[0102] Prepare the amplification system

[0103] Prepare the isothermal nucleic acid amplification system (volume 50 μL) in a 200 μL centrifuge tube according to the following ratio:

[0104]

[0105] The above-prepared amplification system was lyophilized under negative pressure in a freeze dryer to become a powdery amplification system.

[0106] Add polyethylene glycol with a final concentration of 6% (w / v) and a molecular weight of 35,000 to the centrifuge tu...

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Abstract

The invention provides a method for detecting DNA of schistosoma through a fluorescent probe. An amplification system and the DNA of the schistosoma are mixed, isothermal amplification is carried out to obtain an amplified product, and the amplified product is subjected to real-time detection through a fluorescent probe method. The amplification system comprises the components of a Tris buffer solution, potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol, ATP, dNTPs, phosphocreatine single-strand binding protein, recombinase, UvsY protein, exonuclease, the fluorescent probe, DNA polymerase and a schistosoma DNA specific primer. The temperature of isothermal amplification is 35-45 DEG C. The time of isothermal amplification is 5-20 min. The method does not rely on a thermal cycler, operation is easy, the DNA of the schistosoma can be detected at the low temperature (35-45 DEG C), the time used for amplification is short (5-20 min), and the method is suitable for primary and on-site detection.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a method for detecting schistosome DNA with a fluorescent probe. Background technique [0002] Schistosomiasis japonicum is a zoonotic parasitic disease distributed worldwide. It is defined as "one of the six major tropical diseases" by the World Health Organization (WHO) and one of the five major parasitic diseases in my country. Schistosomiasis japonicum is caused by cercariae infecting the ultimate hosts such as humans and animals exposed to infected water. The larva develops in the final host by absorbing the host's nutrition, causing malnutrition in the host; resisting the killing effect of the host's immunity by changing antigens, antigen camouflage, and weakening the host's immunity, and gradually develops into adults and parasitizes in the portal-mesenteric vein system. In the acute phase, the symptoms are fever, hepatomegaly, abdominal pain, diarrhea, and blo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6888C12Q2521/507C12Q2521/319C12Q2522/101C12Q2527/101C12Q2563/107
Inventor 赵松杨坤李伟张键锋羊海涛郭利川刘小曼王智宏应清界
Owner JIANGSU INST OF PARASITIC DISEASES
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