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Universal nucleic acid detection reagent for enteroviruses EV71, CA16 and EV

An EV71 and enterovirus technology, applied in the field of pathogen detection, can solve the problems of poor tolerance, single type of applicable samples, and less coverage of common types of enteroviruses, achieving good sensitivity and improving reaction efficiency.

Active Publication Date: 2020-09-11
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently commercially available fluorescent PCR reagents must be stored frozen at -20°C, and they have low coverage for common types of enteroviruses, poor tolerance to inhibitors such as cholate and heme, and are suitable for a single type of sample or Too few and other issues

Method used

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  • Universal nucleic acid detection reagent for enteroviruses EV71, CA16 and EV
  • Universal nucleic acid detection reagent for enteroviruses EV71, CA16 and EV
  • Universal nucleic acid detection reagent for enteroviruses EV71, CA16 and EV

Examples

Experimental program
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Embodiment 1

[0080] The preparation of embodiment 1 kit

[0081] Design primers and probes based on the conserved sequences of enteroviruses EV71, CA16, and EV viruses:

[0082] EV71 upstream primer: 5'-CACTCACGCCTCTACCAGCAC-3';

[0083] EV71 downstream primer: 5'-AAGAACACAGCGTGTCTCAA-3';

[0084] CA16 upstream primer: 5'-GGRGCYCCRAAACCYACHTCC-3'

[0085] CA16 downstream primer: 5'-TTGRAGRTGCTCNCCGAARG-3'

[0086] EV universal upstream primer: 5'-CCCTGAATGCGGCTAATCC-3'

[0087] EV universal downstream primer: 5'-ATTGTCACCCATAAGCAGCCA-3'

[0088] EV71 probe: 5'-CTTGGAGTGCTGGAACCTTACCTG-3';

[0089] CA16 probe: 5'-CAAGRTTCRGTCCCCTTCATGTCACCAGC-3';

[0090] EV Universal Probe: 5’-AACTCTGCAGCGGAACCGACTACTTTGG-3’

[0091] The PCR reaction solution 1 and the PCR reaction solution 2 of each group are prepared from water and components with the following concentrations:

[0092] Table 1 Reagent formula

[0093]

[0094]

[0095] Reaction solution 1 and reaction solution 2 are packag...

Embodiment 2

[0098] Example 2 sample detection

[0099] Enterovirus EV71, CA16, EV universal type nucleic acid detection kit is used for detecting throat swab, herpes fluid, feces and anal swab unknown sample with the enterovirus EV71, CA16, EV general type described in above-mentioned embodiment 1 -The operation steps of RNA are:

[0100] 1. Reagent preparation

[0101] According to the number of samples to be tested, negative control and positive control, mix in proportion (PCR reaction solution 1 10 μL / person + PCR reaction solution 2 10 μL / person), that is, reaction solution 1 and reaction solution 2 at a volume ratio of 1:1 Mix well to form a PCR-MIX mixture, centrifuge briefly and set aside.

[0102] 2. Sample processing

[0103] 1. Pharyngeal swab and anal swab samples: Put the swab to be tested in 3mL of normal saline, wash it 5-10 times, and the liquid after washing is used as the sample to be tested;

[0104] 2. Stool sample: Take 0.2g in 3mL of normal saline, shake it evenly...

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Abstract

The invention relates to the technical field of pathogen detection, in particular to a universal nucleic acid detection reagent for enteroviruses EV71, CA16 and EV. The universal nucleic acid detection reagent for the enteroviruses EV71, CA16 and EV provided by the invention can tolerate interference of inhibitors, namely cholate and haemachrome due to the addition of an anti-inhibition component;and furthermore, the reagent can be stably stored at 2-8 DEG C and avoids the harsh condition of cryopreservation at -20 DEG C, so that unfreezing is not needed before reaction, and the reaction efficiency is improved. The reagent is suitable for more sample types, such as a throat swab, excrement, a herpes fluid and a cloacal swab when prepared into a kit. A universal primer of the kit covers more serotypes, including coxsackie A, coxsackie B, the enterovirus 71 and an echo virus. The kit has good sensitivity, specificity and repeatability.

Description

technical field [0001] The invention relates to the technical field of pathogen detection, in particular to enterovirus EV71, CA16, EV universal nucleic acid detection reagents. Background technique [0002] Enterovirus (enterovirus, EV) belongs to the genus Enterovirus in the family Picornaviridae. There are many types of enteroviruses that can cause human disease, including human enteroviruses A, B, C, and D groups. There are more than 100 serum Human-to-human enteroviruses are generally susceptible, and different age groups can be infected. Children aged 5 and under are the main ones, and children aged 3 and under have the highest incidence rate, accounting for 85% to 95% of the incidence. Enterovirus enters the upper respiratory tract, throat and intestinal tract as the entry portal, and first proliferates in the local mucosa, pharynx, tonsil and other lymphoid tissues and intestinal Peyer's lymph nodes, and then releases into the blood, forming the first viremia. It sp...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686Y02A50/30
Inventor 李宁高歌高利飞李振红付光宇
Owner AUTOBIO DIAGNOSTICS CO LTD
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