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Primer mixture, kit and detection method for detecting hookworms americana

A primer mixture, technology of hookworm americanum, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of complex operation, expensive equipment, time-consuming, etc., and achieve simple operation and low cost Low, short amplification time effect

Pending Publication Date: 2022-07-29
云南省寄生虫病防治所
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  • Claims
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AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a primer mixture, a kit and a detection method for detecting Ancylostoma americanus, to solve the problem of long time consumption, low sensitivity, complicated operation, expensive instruments and equipment, high requirements for operators, and easy detection in the prior art. Cause problems such as missed detection

Method used

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  • Primer mixture, kit and detection method for detecting hookworms americana
  • Primer mixture, kit and detection method for detecting hookworms americana
  • Primer mixture, kit and detection method for detecting hookworms americana

Examples

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Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Screening of optimal primer sets and optimal reaction conditions

[0040] (1) LAMP amplification specific primers are designed online according to the ITS gene of N. americana, and 3 groups of primers are designed in the present embodiment, as shown in Table 1:

[0041] Table 1

[0042]

[0043]

[0044] (2) Use the hookworm DNA positive plasmid (GenBank accession number: KM891738.1) purchased from Shanghai Poshang Biological Co., Ltd. as the template for LAMP amplification. Of course, those skilled in the art can use the fecal DNA extraction kit QIAamp according to conventional technical means. DNA Stool Mini Kit and routine laboratory techniques were used to extract the egg DNA of N. americana from the samples; a suitable positive plasmid was constructed as a template for LAMP amplification, and the size of the positive plasmid fragment was 600bp.

[0045] (3) Design LAMP amplification system as shown in Table 2:

[0046] Table 2

[0047]

[0048...

Embodiment 2

[0059] Example 2 LAMP and PCR amplification sensitivity test

[0060] According to the calculation formula of plasmid copy number, the original plasmid copy number of hookworm DNA positive plasmid (GenBank accession number: KM891738.1) purchased by Shanghai Poshang Biological Company was calculated as 3×10 10 copies / μl, and serially diluted 10-fold, 3×10 9 copies / μl, 3×10 8 copies / μl, 3×10 7 copies / μl, 3×10 6 copies / μl, 3×10 5 copies / μl, 3×10 4 copies / μl, 3×10 3 copies / μl, 3×10 2 copies / μl, 3×10 1 copies / μl, 1 μl of each was taken as a template for LAMP and PCR amplification, PCR amplification was used as a parallel control experiment, the detection limits of the two were compared, and a negative control N was set.

[0061] (1) LAMP sensitivity test

[0062] 1) The copy number of each template was detected according to the amplification system, optimal primer set, and optimal reaction conditions of Example 1.

[0063] 2) Interpretation of results

[0064] According ...

Embodiment 3

[0074] This embodiment is to detect the specificity of the primer set designed in Example 1 and the detection system constructed, and the process is as follows:

[0075] With reference to Example 1, the feces DNA extraction kit QIAamp DNA Stool Mini Kit and the DNA extraction method of conventional laboratory techniques were used to extract the eggs and negative fecal samples of Taenia taeniae, Ascaris, Amoeba, Fasciola hepatica (without any worm eggs) DNA was used as a template, and the optimal primer set, amplification system, and optimal reaction conditions designed in Example 1 were used for amplification, and the specificity of the primer set was checked, and the N. americana plasmid DNA was used as the standard positive control.

[0076] The sample information from the site is as follows: 17 H. americana positive fecal DNA, 1 Ascaris-positive DNA, 1 Taenia solium-positive DNA, 1 Fasciola hepatica-positive DNA, 1 amoeba-positive DNA, and 2 negative fecal DNA , a total of...

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Abstract

The invention discloses a primer mixture, a kit and a detection method for detecting hookworms americana, the primer mixture comprises: a primer F3.3, the nucleotide sequence of which is as shown in SEQ ID NO: 1; the nucleotide sequence of the primer B3.3 is as shown in SEQ ID NO: 2; the nucleotide sequence of the primer FIP3 is as shown in SEQ ID NO: 3; the nucleotide sequence of the primer BIP3 is as shown in SEQ ID NO: 4; the nucleotide sequence of the primer LF3 is as shown in SEQ ID NO: 5; the nucleotide sequence of the primer LB3 is shown as SEQ ID NO: 6. The invention further provides application of the primer mixture in a kit for detecting the hookworms as well as a detection system and a detection method for the hookworms. The method has the advantages of short amplification time, simplicity in operation, low cost, high sensitivity and good specificity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to the detection technology of hookworm americana. Background technique [0002] American hookworm, nematodes of the family Ancylostomatidae. The adult hookworm parasites in the small intestine of the human body, causing long-term chronic blood loss in the human body, causing hookworm disease (hookworm disease), and patients will experience anemia and anemia-related symptoms. In severe cases, the labor force is significantly reduced, and even life safety is endangered. [0003] For the detection of hookworm Americana infection, the modified Kato thick smear method recommended by the World Health Organization is currently mainly used. This method is economical and convenient, and the diagnosis can be confirmed by observing the eggs under the microscope; other detection methods include polymerase chain reaction, immunology and other methods. However, these methods have...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2531/119
Inventor 杨亚明郭建妮蔡璇吴方伟李奔福严信留彭佳字金荣王正青徐倩李建雄
Owner 云南省寄生虫病防治所
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