SNP molecular marker ITS296 for detecting dog-derived Ceylon hookworms and ancylostoma caninum, primer and application thereof

A technique of hookworm of ceylon and molecular marker is applied in the field of hookworm disease diagnosis and detection, which can solve the problems of high cost, inability to detect a large number of samples at the same time, complicated operation, etc., and achieve the effect of completely consistent accuracy and completely consistent sequencing results.

Active Publication Date: 2018-04-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned technologies all have certain limitations. For example, some of them cannot detect a large number of samples at the same time, and some of them are cumbersome and expensive.

Method used

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  • SNP molecular marker ITS296 for detecting dog-derived Ceylon hookworms and ancylostoma caninum, primer and application thereof
  • SNP molecular marker ITS296 for detecting dog-derived Ceylon hookworms and ancylostoma caninum, primer and application thereof
  • SNP molecular marker ITS296 for detecting dog-derived Ceylon hookworms and ancylostoma caninum, primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Identification of Ancylostoma Ceylonium from dogs and Ancylostoma caninum

[0049] (1) First refer to the ITS sequences of Hookworm canine (LC177192.1) and Hookworm Ceylon (KM066110.1) downloaded from NCBI, and design a set of T based on a SNP site ITS296 of ITS1. m -shift PCR primers (2 forward specific primers and 1 common reverse primer), see Table 1.

[0050] Table 1 T based on ITS1 sequence SNP site ITS296 m -shift primer

[0051]

[0052] Establish T according to the above primers m -shift PCR reaction system (Table 2) and reaction conditions:

[0053] Table 2 T m -shift PCR reaction system

[0054]

[0055] Reaction conditions:

[0056]

[0057] (2) T based on SNP site ITS296 m -shift primers for the standard plasmids of Hookworm Ceylon (AceP) and Hookworm (AcaP) m -shift standard curve see figure 1 . The results showed that the T based on SNP site ITS296 m The -shift method can distinguish Ceylon hookworm from dog hookworm. Through the analysis of Rotor-Gene Q...

Embodiment 2

[0058] Example 2.T m -shift method stability test

[0059] To evaluate the established T based on SNP locus ITS296 m -The stability of the shift method, the test carried out reproducibility testing on the constructed two known plasmid standards. Each sample is tested 7 times in batches, and 3 times between batches, qPCR-T m -shift reaction system and cycle parameters are the same as above. The test results showed that in the primers of ITS296, Ancylostoma canis and Ancylostoma T m The CV of the value is 0.07% and 0.13% respectively (Table 3), so the T m The -shift molecular detection method has good intra-batch and inter-batch reproducibility.

[0060] Table 3 Repeatability test results

[0061]

Embodiment 3

[0062] Example 3.T m -shift method sensitivity test

[0063] To detect established T based on SNP site ITS296 m -Sensitivity of the shift method. Dilute the prepared two plasmid standards by 10-fold ratio respectively, and press 1:10 to 1:10 8 Concentration is detected, qPCR-T m -shift reaction system and cycle parameters are the same as above. The results showed that the dilution of H. canis and H. Ceylon samples was as low as 1:10 7 (I.e. 5.33×10 -6 ng / μL and 5.03×10 -6 ng / μL), T m The -shift method can still distinguish the two (see Table 4).

[0064] Table 4 The T of different concentrations of Ancylostoma caninum and Ancylostoma Ceylonum m value

[0065]

[0066] Note: "-" means that the target peak cannot be detected

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Abstract

The invention discloses an SNP molecular marker ITS296 for detecting dog-derived Ceylon hookworms and ancylostoma caninum, a primer Tm-shift and application thereof. The SNP molecular marker ITS296 ispositioned at the 296th-position basic group T or G as shown in SEQ ID NO:1, the position with the basic group T is the Ceylon hookworms, and the position with the basic group G is the ancylostoma caninum. The primer designed on the basis of the SNP site ITS296 can accurately identify the Ceylon hookworms and the ancylostoma caninum; the CV of the Tm values of the Ceylon hookworms and the ancylostoma caninum are 0.13 percent and 0.07 percent correspondingly; when the dilution degree of the Ceylon hookworms and the ancylostoma caninum is less than 1:107, the Ceylon hookworms and the ancylostoma caninum still can be differentiated; the detection results of 10 known hookworm to-be-detected DNA samples are completely consistent with those of the known specifies; the detection rate of the clinical samples is obviously higher than that of microscopic examination, and the accurate rate and the sequencing result are completely consistent. The technology can be applied to species identification of the dog-derived hookworms, molecular epidemiological investigation and risk evaluation of hookworm disease outbreak.

Description

Technical field [0001] The invention belongs to the technical field of hookworm diagnosis and detection, and specifically relates to a SNP molecular marker ITS296, T m -shift primer and its application. Background technique [0002] Ancylostome (Ancylostome) is a common parasite distributed worldwide, which can cause serious harm to the health of dogs, cats and people. Hookworms that parasitize dogs include Ancylostoma caninum, A. ceylanicum, A. braziliense and Uncinaria stenocephala (Chilton and Gasser, 1999). According to the survey, the hookworms infected by Chinese dogs in recent years are mainly Ceylon hookworm and canine hookworm (Liu et al., 2015). At present, Ceylon hookworm has become the second largest type of hookworm infecting humans in Asia (Traub, 2013), especially in Southeast Asian countries, such as China (Chen et al., 2012; Liu et al., 2014), Japan (Kaya et al., 2016), Malaysia (Nguiet al., 2012), Laos (Conlan et al., 2012), Thailand (Traub et al., 2008), Indi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2527/107
Inventor 李国清傅叶琪王明威潘伟达石先利胡伟刘远佳
Owner SOUTH CHINA AGRI UNIV
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