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Kit and a detection method for high-throughput detection of intestinal protozoa based on honeycomb chip

A high-throughput, kit-based technology, applied in the determination/testing of microorganisms, biochemical equipment and methods, and resistance to vector-borne diseases, etc., to achieve the effects of reducing the incidence of false positives, good specificity, and improving detection efficiency

Pending Publication Date: 2021-10-19
中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the detection of intestinal protozoa, the single tube of LAMP is still used to detect pathogens, and the related gene chips for intestinal protozoa have not been prepared yet

Method used

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  • Kit and a detection method for high-throughput detection of intestinal protozoa based on honeycomb chip
  • Kit and a detection method for high-throughput detection of intestinal protozoa based on honeycomb chip
  • Kit and a detection method for high-throughput detection of intestinal protozoa based on honeycomb chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0172] Example 1 DNA extraction of different strains.

[0173] (1) Positive samples of Cryptosporidium, Giardia, Amoeba histolytica, and Microsporidium were isolated from the laboratory and stored in potassium dichromate at 4°C.

[0174] (2) On-site samples were collected from diarrhea patients and animal feces samples in hospitals in Shanghai, Jiangsu and other places.

[0175] I: Fecal DNA extraction kit was purchased from Qiagen, USA, and PCR mix was purchased from Bio-Serve.

[0176] II: The nucleic acid of the sample was carried out in accordance with the "Technical Plan for Diarrheal Syndrome Detection" for molecular detection of related pathogens, and the species of the sample was confirmed by sequencing and comparison.

Embodiment 2

[0177] Example 2 Independent design and screening of detection primers for 5 kinds of intestinal protozoa

[0178] (1) Based on bioinformatics analysis, different target genes were used for target design.

[0179] (2) Primer design.

[0180] (3) Primer screening

[0181] Screening strategy: perform literature search for each strain first, and select literature-reported primers for verification. If the primers are valid, they will be used as spare primers for the chip; The possible primers were verified one by one to obtain effective specific amplification primers.

[0182] Among them, the sequences of Cryptosporidium human, Cryptosporidium turkey, Giardia, and amoeba were independently designed and synthesized; the general primers for Cryptosporidium (associated with 2 loop primers), Cryptosporidium parvum, and Cryptosporidium ansei Literature primers were used; one primer from Microsporidia was designed as a degenerate primer. See below for primers:

[0183] Group 1, Cry...

Embodiment 3

[0232]Example 3 Plasmid construction

[0233] Download the sequence of each strain according to the NCBI database. After comparing the sequences of different isolates of the same strain, select the appropriate region for template synthesis; construct a plasmid, which is consistent with the target sequence after sequencing;

[0234] A total of 8 template plasmids:

[0235] (1) CRY-403bp, the nucleotide sequence is shown in SEQ ID NO.39;

[0236] (2) Ch-244bp, the nucleotide sequence is shown in SEQ ID NO.40;

[0237] (3) Cp-221bp, the nucleotide sequence is shown in SEQ ID NO.41;

[0238] (4) Cm-408bp, the nucleotide sequence is shown in SEQ ID NO.42;

[0239] (5) Ca-230bp, the nucleotide sequence is shown in SEQ ID NO.43;

[0240] (6) GD-810bp, the nucleotide sequence is shown in SEQ ID NO.44;

[0241] (7) E.his-266bp, the nucleotide sequence is shown in SEQ ID NO.45;

[0242] (8) EnW-310bp, the nucleotide sequence is shown in SEQ ID NO.46.

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Abstract

The invention discloses a kit and a detection method for high-throughput detection of intestinal protozoa based on a honeycomb chip, which are integrated on a honeycomb chip by adopting a microfluidic system and used for detecting cryptosporidium, giardia, microsporidium and histolytic amoeba. The kit comprises any one or a combination of any more of the following eight groups of primers: a specific primer of cryptosporidium; a specific primer of human cryptosporidium; a specific primer of cryptosporidium parvum; a specific primer of cryptosporidium turkey; a specific primer of cryptosporidium andersoni; a specific primer of giardia giardia; and a specific primer of amoeba. The invention relates to a specific primer of enterosporidia. According to the invention, the tedious steps of carrying out PCR detection for many times are solved, the effects of simultaneously detecting a plurality of genes and determining pathogens can be achieved by one reaction, the workload is greatly reduced, the detection time is shortened, and the detection cost is reduced.

Description

technical field [0001] The invention relates to the technical fields of agriculture and animal quarantine, in particular to a detection method for Cryptosporidium, Giardia, Microsporidium and Amoeba histolytica, and in particular to a high-throughput detection method for a variety of intestinal bacteria based on a honeycomb chip. Multiplex PCR detection method and kit for Daoplasma. Furthermore, the present invention relates to specific primers for the simultaneous detection of Cryptosporidium, Giardia, Microsporidia and Amoeba histolytica. Background technique [0002] Cryptosporidium, Giardia lamblia (referred to as Giardia), Enteromicrosporidium pichia and Amoeba histolytica are zoonotic new intestinal protozoa, mainly transmitted by water, causing consumptive diarrhea. It is an important public health problem facing the world, and it is also one of the most difficult problems faced by mankind so far; it can break out and cause serious public health emergencies. Human i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6893C12Q1/6844C12Q2600/16C12Q2531/119C12Q2537/143C12Q2565/629Y02A50/30
Inventor 姜岩岩曹建平沈玉娟袁忠英
Owner 中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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