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SNP molecular marker its71, primers and applications for detecting Ancylostoma ceylonii and Ancylostoma canis

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

Active Publication Date: 2020-04-17
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 its71, primers and applications for detecting Ancylostoma ceylonii and Ancylostoma canis
  • SNP molecular marker its71, primers and applications for detecting Ancylostoma ceylonii and Ancylostoma canis
  • SNP molecular marker its71, primers and applications for detecting Ancylostoma ceylonii and Ancylostoma canis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1. The identification of canine Ancylostoma ceyloni and Ancylostoma canis

[0049] (1) First, refer to the ITS sequences of Ancylostoma caninum (LC177192.1) and Ancylostoma ceylonum (KM066110.1) downloaded from NCBI, and design a set of T 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 ITS71 m -shift primer

[0051]

[0052] According to the above primers to establish T m The reaction system (Table 2) and reaction conditions of -shift PCR:

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

[0054]

[0055] Reaction conditions:

[0056]

[0057] The melting process is 70-95°C, 0.5°C / s.

[0058] (2) T based on SNP site ITS71 m -shift primers for AceP and AcaP standard plasmids m -shift standard curve see figure 1 . The results showed that the T based on the SNP site ITS71 m The -shift method can distinguish Ancylostoma ceyloni from Ancylostoma canis...

Embodiment 2

[0059] Example 2.T m Stability test of -shift method

[0060] To evaluate the established SNP site ITS71-based T m The stability of the -shift method was tested for the repeatability of the two known plasmid standards constructed. Each sample was tested 7 times within a batch and 3 times between batches. qPCR-T m -shift reaction system and cycle parameters are the same as above. The test results showed that among the primers of ITS71, the T m The coefficients of variation (CV) of the values ​​are 0.15% and 0.09% respectively (Table 3), so the T m The intra-assay repeatability and inter-assay repeatability of -shift molecular detection method were both good.

[0061] Table 3 Repeatability test results

[0062]

Embodiment 3

[0063] Example 3.T m Sensitivity test of -shift method

[0064] To detect the established SNP site ITS71-based T m The sensitivity of the -shift method is to dilute the two prepared plasmid standards by 10 times, according to the ratio of 1:10 to 1:10. 8 Concentration detection, qPCR-T m -shift reaction system and cycle parameters are the same as above. The results showed that when the sample dilution of Ancylostoma caninum and Ancylostoma ceylonis 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).

[0065] Table 4 primers to different concentrations of Ancylostoma caninum and Ancylostoma ceyloni T m value

[0066]

[0067] Note: "-" indicates that the target peak cannot be detected

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Abstract

The invention discloses an SNP molecular marker ITS71 for detecting dog-derived Ceylon hookworms and ancylostoma caninum, a primer Tm-shift and application thereof. The SNP molecular marker ITS296 ispositioned at the 71th-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 ITS71 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 0.09 percent and 0.15 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 diagnosis and detection of hookworm disease, in particular to a SNP molecular marker ITS71 and T m -shift primers and their applications. Background technique [0002] Hookworm (Ancylostome) is a common parasite distributed worldwide, which can cause serious harm to the health of dogs, cats and humans. 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 dogs in China in recent years are mainly Ancylostoma ceylonii and Ancylostoma canis (Liu et al., 2015). Currently, Ancylostoma ceylonii has become the second largest species of hookworms 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 (Ngui et al., 2012), Laos (Conlan et al., 2012), Thailand (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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