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Multiple real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for simultaneously detecting three types of echinococcus

A real-time fluorescent quantitative and Echinococcus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of harmfulness to operators and the environment, complicated operation, and low sensitivity , to achieve the effect of good quantitative linear range, simple operation and high sensitivity

Active Publication Date: 2021-05-14
QINGHAI ACAD OF ANIMAL SCI & VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, in my country, the distinction between Echinococcus multilocularis (Em), Echinococcus granulosus (Eg) and Echinococcus shiquense (Es) is mainly through microscopic examination of parasite and egg morphology, as well as molecular biology and immunology. These methods are harmful to the operator and the environment, or the operation is complicated, or the sensitivity is low, or there are problems such as missed detection, false positive, etc.

Method used

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  • Multiple real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for simultaneously detecting three types of echinococcus
  • Multiple real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for simultaneously detecting three types of echinococcus
  • Multiple real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for simultaneously detecting three types of echinococcus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The sample is a positive plasmid standard containing target fragments of Em, Eg and Es, and is amplified by PCR using a Roche kit (RocheProbe PCRKit).

[0053] PCR amplification system: sample template 2 μl, 10 μm / L Em g s-F 1.5 μl, 10 μm / L Em g s-R 1.5 μl, 10 μm / L Em probe 0.2 μl, 10 μm / L Eg probe 0.2 μl, 10 μm / L Es probe 0.2μl, 2×ProbeMasterMix 10μl, RNA free H 2 O 4.4 μl.

[0054] Em g s-F: 5'-TTGTTTGCTATGTTTTCTATAGTGT-3'; SEQ ID NO.1;

[0055] Em g s-R: 5'-TCTTCACATCYAACCCAACAGT-3'; SEQ ID NO.2;

[0056] Among them, Y=C / T;

[0057] The probes used to detect Em are:

[0058] Emprobe: 5'-TTTAGGGAGTAGTGTTT-3'; SEQ ID NO.3;

[0059] The fluorescent group labeled at the 5' end is FAM, and the quencher group labeled at the 3' end is BHQ1;

[0060] The probes used to detect Eg are:

[0061] Eg probe: 5'-TTTGGGTAGCAGGGTT-3'; SEQ ID NO.4;

[0062] The fluorescent group labeled at the 5' end is VIC, and the quencher group labeled at the 3' end is BHQ1;

[0063] The p...

Embodiment 2

[0076] Embodiment 2 specificity test

[0077] Genomic DNA of parasites (Tenna polycephala, Taenia saginata, Taenia asiatica, Dipyridium canis, Taenia alveolar, Toxocara canis, Fasciola hepatica, Taenia lentiformis, Taenia centrum, Cryptosporidium canis) worm) and positive plasmid standard substance as template, carry out specificity test (test process is the same as embodiment 1), the results are shown in Figure 8 ; Figure 8 The results show that only Echinococcus multilocularis Em, Echinococcus granulosus Eg and Echinococcus shiqu Es positive samples have amplification curves, which are positive, and the rest of the control samples are negative, indicating that The method has good specificity.

Embodiment 3

[0079] The Ct values ​​of the triple real-time quantitative method and the single real-time quantitative method were compared, and the results are shown in Table 3.

[0080] Table 3 Comparison of Ct values ​​between the triple real-time quantitative method and the single real-time quantitative method

[0081]

[0082] Note: (triple: a pair of universal primers plus three probes; singleplex: a pair of universal primers plus a corresponding probe) for the detection of the same sample 1, sample 2, and sample 3.

[0083] The results in Table 3 show that the triple PCR simultaneous detection and the single PCR detection of the same sample have little change in the detection value, and the detection results are reliable.

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Abstract

The invention discloses multiple real-time fluorescent quantitative PCR (polymerase chain reaction) primers, probes and a kit for simultaneously detecting three types of echinococcus, and belongs to the technical field of parasitic pathogen detection. According to the multiplex real-time fluorescent quantitative PCR primer and the probe for simultaneously detecting the three kinds of echinococcus, the three kinds of pathogens of echinococcus multilocularis, echinococcus granulosus and echinococcus shigii are simultaneously detected, the detection efficiency is greatly improved, and the detection time and the detection cost are saved. According to the present invention, the specificity is strong, no non-specific amplification is performed on other common related parasite gene samples, and the sensitivity is high and is as high as 10 copies / [mu] l;.

Description

technical field [0001] The invention relates to the technical field of detection of parasitic pathogens, in particular to multiple real-time fluorescence quantitative PCR primers and probes and a kit for simultaneously detecting three kinds of Echinococcus. Background technique [0002] Hydatid disease, also known as echinococcosis, is an important zoonotic parasitic disease caused by tapeworm larvae of the genus Echinococcus. One of the neglected tropical diseases. Humans or rodents are infected by eating water or food contaminated by eggs. In my country, cystic echinococcosis (Cysticechinococcus) and multilocular The prevalence of Alveolar echinococcosis caused by Echinococcus multilocularis (E. multiloculari, Em) is mainly distributed in northwest pastoral areas such as Qinghai, Xinjiang, Gansu, Tibet and Sichuan. Echinococcosis is mainly distributed in North America, Europe and Asia in the cold regions and tundra in the high latitudes of the northern hemisphere, such as...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 张学勇简莹娜
Owner QINGHAI ACAD OF ANIMAL SCI & VETERINARY MEDICINE
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