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Real-time fluorescence multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for simultaneously detecting human coronaviruses 229E, OC43, NL63 and HKU1

A RT-PCR, coronavirus technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as high sensitivity real-time quantification, which is not conducive to early diagnosis of diseases

Active Publication Date: 2017-05-10
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that it has a hysteresis, so it is not conducive to the early diagnosis of the disease
At present, there is no kit with simple operation, high sensitivity, real-time quantification, and the ability to simultaneously detect multiple coronaviruses on the market

Method used

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  • Real-time fluorescence multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for simultaneously detecting human coronaviruses 229E, OC43, NL63 and HKU1
  • Real-time fluorescence multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for simultaneously detecting human coronaviruses 229E, OC43, NL63 and HKU1
  • Real-time fluorescence multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for simultaneously detecting human coronaviruses 229E, OC43, NL63 and HKU1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] Primer design and screening

[0144] Download the existing sequences of human coronavirus 229E, OC43, NL63 and HKU1 in Genebank, select the M gene of 229E, the N gene of OC43, the N gene of NL63 and the N gene of HKU1 as the target genes for detection, using MEGA software Multiple sequence alignments were carried out to find out the respective conserved regions.

[0145]According to the comparison results, 12 pairs of primers were designed in the conserved region of each target gene using "NCBI / Primer-BLAST", including 3 pairs of primers for amplifying the M gene of 229E (SEQ ID NO.: 1, 2, 19-22) , 4 pairs of primers (SEQ ID NO.: 9, 10, 23-28) for amplifying the N gene of OC43, 1 pair of primers for amplifying the N gene of NL63 (SEQ ID NO.: 5, 6) and 4 pairs of primers for amplifying the N gene of NL63 Primers for increasing the N gene of HKU1 (SEQ ID NO.: 7, 8, 29-34). All primers are analyzed by "Primer-BLAST" to ensure that there is no cross-homology with other nu...

Embodiment 2

[0156] Modification of OC43 primer

[0157] From the results of melting curve analysis of the four templates amplified by the best primer combination in Example 1, it can be seen that the difference in Tm between 229E and OC43 is less than 1°C. In the absence of a better primer scheme , try to modify the primers of OC43 as follows:

[0158] Independently select a GC-rich sequence containing 9 bases: ATCCCGGGG, use the "Scrambled DNA Sequence Tool" in the "Sequence Processing Online Toolkit (SMS)" to generate a series of sequences with the same base composition as the fragment For fragments with different base sequencing, try to add the 5' ends of the OC43 amplification primers (SEQ ID NO: 9, 10) selected in Example 1, respectively.

[0159] All modified primer sequences were analyzed using the software "Vector NTI", and the modified primers that formed primer-dimers and less hairpin structures were screened out, and the optimized modified sequences were as follows:

[0160] ...

Embodiment 3

[0165] RNA in vitro transcription

[0166] The T7 promoter sequence: TAATACGACTCACTATAG was added to the 5' ends of the upstream primers of the four viruses (see sequence table SEQ ID NO.: 1, 5, 7 and 9), and the modified primer sequence was named T7-229E-F ( SEQ ID NO.:11), T7-OC43-F (SEQ ID NO.:12), T7-NL63-F (SEQ ID NO.:13), T7-HKU1-F (SEQ ID NO.:14).

[0167] The four viruses were amplified using the above primers in combination with their corresponding downstream primers (see SEQ ID NO.: 2, 6, 8, 10 in the sequence listing).

[0168] After the amplified product is recovered and purified, it can be used as a template for in vitro transcription.

[0169] Promega's "RiboMAX Large Scale RNA ProductionSystem-T7" kit was used for in vitro transcription, and the reaction process used the method provided by the reagent manufacturer. The concentration and purity of RNA products transcribed in vitro were measured using Nanodrop, and the unit was converted into copies / μL. To avoi...

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Abstract

The invention provides a primer for real-time fluorescence multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and a related kit as well as a method for simultaneously detecting human coronaviruses 229E, OC43, NL63 and HKU1 by using the primer. By use of a real-time fluorescence multi-RT-PCR detection method of the primer, the four coronaviruses can be simultaneously detected by the dissolving temperature of an amplified product; the real-time fluorescence multi-RT-PCR detection method has the advantages of simple operation, convenience, quickness, good specificity and high sensitivity, and is suitable for clinical detection and laboratory detection.

Description

technical field [0001] The invention relates to the technical field of biological detection of viruses, in particular to primers and related kits used in real-time fluorescent multiple RT-PCR for simultaneous detection of human coronavirus 229E, OC43, NL63 and HKU1. Background technique [0002] Human coronavirus (HCoV) is a single-stranded positive-sense RNA virus with a mantle, belonging to the family Coronaviridae. Because the corona-like outer crown can be seen under the electron microscope, it is called a coronavirus. At present, there are 6 types of human coronaviruses that have been discovered. Four of these human coronaviruses (229E, OC43, NL63, and HKU1) are currently common in the population and are associated with most human respiratory diseases, such as common colds and high incidences of pneumonia and bronchitis. Coronavirus infection is distributed in many regions of the world. The virus is excreted through respiratory secretions, transmitted through oral flu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2537/143C12Q2561/113C12Q2563/107
Inventor 张弛宇张亚楠蓝柯
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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