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Special primer capable of quickly detecting human coronavirus NL63 as well as kit and application method thereof

A coronavirus, human detection technology, applied in the fields of molecular biology and virology, to achieve specific results in the detection process

Inactive Publication Date: 2015-09-16
江苏国际旅行卫生保健中心无锡分中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the detection of HCoV-NL63 gene by LAMP method at home and abroad

Method used

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  • Special primer capable of quickly detecting human coronavirus NL63 as well as kit and application method thereof
  • Special primer capable of quickly detecting human coronavirus NL63 as well as kit and application method thereof
  • Special primer capable of quickly detecting human coronavirus NL63 as well as kit and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 special primer design

[0031] The 32 currently known full-length genome sequences of HCoV-NL63 registered on GenBank were analyzed by Clustal software, and the domestic strain (GenBank accession number JX104161) was used as the reference sequence, and PrimerExplorer4.0 software was used to target HCoV-NL63. Eight primers were designed for the coding region of NL63 nucleocapsid (N) gene, namely F3, F2, F1, B1, B2, B3, LF, and LB. Among them, F3 and B3 are two outer primers, FIP (composed of F1 and F2) and BIP (composed of B1 and B2) are two inner primers, and LF and BF are two loop primers. See the table below for primer information:

[0032] F3: CATTTTACATGCCTCTTTTGG

[0033] B3: CCTTATGAGGTCCAGTACC

[0034] FIP: TCCAATAACCAATCTGCTCATCTTTTGATAAGGCACCATATAGGGT

[0035] BIP: GTTCAAGAGCGTTGGCGTATAAAATGAACTTTAGGAGGCAAAT

[0036] LF: GGGACAAGATTCCTGGGAATG

[0037] BF: CAGGGGGCAACGTGTTG.

Embodiment 2

[0038]Example 2 Primer Specificity Test

[0039] Four common human coronaviruses, HCoV-NL63, HCoV-OC43, HCoV-229E and HCoV-HKU, as well as influenza A virus (H3N2) and influenza B virus (Flu B), Norovirus (Norovirus) genome was used as a template respectively, and deionized water was used as a negative control, and the following 25 μL reaction system was established according to the RT-LAMP kit: 12.5 μL of 2x reaction buffer (RM), six specific primers ( F3, B3, FIP, BIP, LF, BF) 1 μL each (primer concentrations are 5 μM, 5 μM, 40 μM, 40 μM, 20 μM, 20 μM), visual fluorescence reagent (calcein) 1 μL, enzyme solution (EM) 1 μL, virus 4.5 μL of RNA template was mixed evenly and placed in an LA-500 Loopamp instrument for constant temperature reaction at 63°C for 60 minutes, then at 80°C for 5 minutes to terminate the reaction and observe the color change in the reaction tube. The result is shown in the figure below:

[0040] from figure 1 It can be seen that only in the reaction...

Embodiment 3

[0041] Example 3 Primer Sensitivity Test

[0042] Use a spectrophotometer to quantify HCoV-NL63 viral RNA, and estimate each microliter according to the formula (copy / μL=(6.02x1023)xconcentration (ng / μL)x10-9 / (target fragment base number x340)) Viral RNA copy number. The RNA was serially diluted to 10 by 10-fold dilution 0 times, 10 1 times, 10 2 times, 10 3 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times, 10 8 times. Establish the following 25 μL RT-LAMP reaction system: 12.5 μL of 2x reaction buffer (RM), 1 μL of each of the six specific primers (F3, B3, FIP, BIP, LF, BF) (primer concentrations were 5 μM, 5 μM, 40 μM, 40 μM, 20 μM, 20 μM), 1 μL of visual fluorescence reagent (calcein), 1 μL of enzyme solution (EM), 5 μL of viral RNA template diluted in each ratio, mix well and place in LA-500 Loopamp instrument for constant temperature reaction at 63°C for 60 min, and Observe the change of reaction turbidity value in real time. After the end of the reaction...

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Abstract

The invention discloses a special primer capable of quickly detecting human coronavirus NL63 as well as a kit and an application method thereof. The special primer consists of six primers, which are respectively F3, FIP (Feline Infectious Peritonitis), BIP (Binding Ig Protein), B3, LF (Lactoferrin) and LB (Liquid-Phase Basicity), wherein the F3 and the B3 are two outer primers, the FIP is an inner primer consisting of the F1 and the F2 and an inner primer BIP consisting of the B1 and the B2, and the LF and the BF are two annular primers; a nucleotide sequence is as representn below: CATTTTACATGCCTCTTTTGGB3; CCTTATGAGGTCCAGTACCFIP; TCCAATAACCAATCTGCTCATCTTTTGATAAGGCACCATATAGGGTBIP; GTTCAAGAGCGTTGGCGTATAAAATGAACTTTAGGAGGCAAATLF; GGGACAAGATTCCTGGGAATGBF; CAGGGGGCAACGTGTTG.

Description

technical field [0001] The invention belongs to the fields of molecular biology and virology, and more specifically relates to a special primer and kit for rapid detection of human coronavirus NL63 (Human coronavirus NL63, HCoV-NL63) gene and a method of use thereof. Background technique [0002] Human coronavirus NL63 (Human coronavirus NL63, HCoV-NL63) is a member of the family Coronaviridae and the genus Coronavirus. 229E belongs to the α subgroup of the genus Coronaviridae. Since HCoV-NL63 was first isolated and identified from nasopharyngeal aspirates of a child with acute respiratory infection in the Netherlands in 2004, its molecular epidemiological studies have shown that the virus is prevalent in many countries and regions, showing a global distributed. The virus is one of the six important human coronaviruses known so far, and its average detection rate in acute respiratory virus infections is 3.7%, mainly infecting infants and adults with weakened or deficient i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/70C12Q1/701C12Q2531/119
Inventor 耿合员汪圣强肖雨张汀顾高浪谢晓谦谭文杰苏川
Owner 江苏国际旅行卫生保健中心无锡分中心
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