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Schistosoma japonicum RPA molecular detection method

A technology for molecular detection and schistosomiasis, applied in the field of temperature amplification detection, can solve problems such as difficult and rapid detection, and achieve the effects of early diagnosis and early warning, easy reaction conditions, and satisfactory reaction conditions

Active Publication Date: 2019-07-19
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Nucleic acid amplification is the basic step in genetic information analysis experiments. Polymerase chain reaction (PCR) amplification technology has shown excellent detection performance in the application of pathogen identification, gene mutation and genetic analysis, but there are certain limitations. Limitations, the reaction requires temperature-controlled equipment for denaturation and annealing steps, RNA samples need to undergo complex processing such as reverse transcription, and professional technicians are required to operate, so it is difficult to be widely used in on-site rapid detection

Method used

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  • Schistosoma japonicum RPA molecular detection method

Examples

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Embodiment 1

[0044] Example 1: Detection of Schistosoma japonicum Adult Samples

[0045] 1. Extraction of total DNA from adults of Schistosoma japonicum

[0046] Tissue DNA Kit ( Genomic DNA Purification Kit, purchased from Promega Company, catalog number A7280) extracts the total DNA of Schistosoma japonicum. Specific operation: Place a pair of male and female adult Schistosoma japonicum in 400 μL of lysate (200 mM EDTA; 40 Mm Tris-HCl; 1% SDS and 0.5 mg / ml proteinase K) in a constant temperature water bath at 37 ° C for 12 h; use a DNA purification kit ( DNA Clear-up System (purchased from Promega, Cat. No. A7280) was used to purify the lysate after water bath, and finally dissolve the DNA with 40-50 μL Elution Buffer.

[0047] 2. Isothermal Amplification of Recombinase Polymerase

[0048] 2.1 Design specific primers and probes according to the partial sequence of Schistosoma japonicum SjCHGCS19 gene (as shown in SEQ ID NO: 1) with the reported accession number FN356221.1:

[0049...

Embodiment 2

[0056] Embodiment 2: RPA detection method specificity test

[0057] Trichostrongylus serpentine, Haemonchus contortus, Fasciola hepatica, Anteroposterior and posterior Dischiflumus, Pancreatica pancreatum, Osternematode used in the specificity test, the above samples are carried out DNA extraction by the method of embodiment 1, RPA Isothermal amplification, and detection of the amplified product, the results are shown in figure 2 and Table 1.

[0058] figure 2 Only test strip 1 has a positive reaction, which is shown as a purple test strip D in the test area; the other 6 parasites corresponding to test strips 2-6 all have negative reactions, which is shown as no test strip.

[0059] Table 1 Specificity tests for RPA detection of Schistosoma japonicum

[0060] Test No. name source Amplification result 1 Schistosoma japonicum Nanjing Center for Disease Control and Prevention + 2 Haemonchus contortus Sampling collection in Tianmen City, Hubei...

Embodiment 3

[0062] Embodiment 3: Sensitivity test of RPA detection method

[0063] Genomic DNA extracted from a single pair of adults of Schistosoma japonicum (the DNA concentration measured by Thermo SCIENTIFIC MANODROP 2000 Spectrophotometer was 40ng / ul) was used as a template to detect the sensitivity of the RPA amplification method of Schistosoma japonicum. Specific steps are as follows:

[0064] 1. The method in Example 1 extracts a single pair of Schistosoma japonicum DNA templates.

[0065] 2. Dilute the extracted genomic DNA with ddH 2 O were diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 、10 -11 、10 -12 Times, for the template DNA PCR and RPA amplification respectively.

[0066] 3. PCR method to amplify the target fragment: amplify the fragment of the SjCHGCS19 gene of Schistosoma japonicum with specific primers F1 and R1 of Schistosoma japonicum,

[0067] Forward primer F1: 5'-CAGCGTAGACAGGAGATCAC-3';

[0068] Reverse primer R1: ...

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Abstract

The invention discloses a schistosoma japonicum RPA molecular detection method. The method comprises the following steps: 1) designing a pair of specific primers and a probe according to a sequence ofa schistosoma japonicum SjCHGCS19 gene, and a nucleotide sequence of the schistosoma japonicum SjCHGCS19 gene is shown in SEQ ID NO: 1; 2) extracting the total DNA in a sample to-be-tested, and performing an isothermal amplification reaction of the recombinase polymerase; and 3) detecting an amplified product. The method has high specificity and sensitivity for the detection of schistosoma japonicum, and can detect schistosoma japonicum by sampling feces or whole blood in a short time after the infection of the animal, and facilitates early diagnosis and early warning of schistosoma japonicumas well as early laboratory identification and screening.

Description

technical field [0001] The invention belongs to the technical field of parasite molecular detection, in particular to a recombinase polymerase isothermal amplification (RPA) detection method of Schistosoma japonicum. [0002] technical background [0003] Schistosoma japonicum is a zoonotic parasitic Heterosoma japonicum. Schistosoma japonicum has a complex life history, widely distributed intermediate hosts, and a variety of transmission or conservation hosts. It seriously endangers the health of humans and animals around the world and affects the development of animal husbandry in various countries. It is one of the five major parasitic diseases that need to be controlled for a long time. The disease mechanism of Schistosoma japonicum has not been fully understood, but studies have found that egg deposition, worm body stimulation, protein antigen secreted by miracidium glands and other pathogenic factors can cause various lesions at the parasitic site (Garcia EG, Mitchell G...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/507C12Q2531/119
Inventor 胡敏朱涛杨新李雪松米坤刘璐
Owner HUAZHONG AGRI UNIV
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