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Kit and method for rapidly detecting human bocavirus based on isothermal amplification

An isothermal amplification, Boca virus technology, applied in the field of molecular biology, to achieve the effect of simple operation, good specificity and rapid diagnosis

Pending Publication Date: 2021-06-01
NINGBO MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there is still no report on the use of RPA technology to detect human bocavirus

Method used

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  • Kit and method for rapidly detecting human bocavirus based on isothermal amplification
  • Kit and method for rapidly detecting human bocavirus based on isothermal amplification
  • Kit and method for rapidly detecting human bocavirus based on isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Selection and preparation of targeting sequences

[0031] Find the published human bocavirus NP1 gene sequence from NCBI, and use the nucleotide sequence between positions 10-279 of the human bocavirus NP1 gene as a template through multiple alignments. The template nucleotide sequence was provided by Shanghai Sangon Biotechnology Synthesized by Engineering Technology Services Co., Ltd. and integrated into the EcoRI site of pBluescriptⅡSK(+) of the plasmid, then transformed the recombinant plasmid into E. coli TOP 10, and inoculated it into LB medium supplemented with 100 μg / mL ampicillin;

[0032] After incubating at 37° C. for 8 h, the plasmid was extracted according to the instructions of the plasmid mini-prep kit (TIANprep Mini Plasmid Kit, purchased from TIANGEN, catalog number DP103). The concentration of the extracted plasmid was measured by ultra-micro spectrophotometer to be 1024ng / μL, and then the plasmid was cleaved. The enzyme digestion system: DN...

Embodiment 2

[0033] Example 2: Design and optimization of primers

[0034] In this example, a series of RPA primers were designed for the conserved region of the human bocavirus NP1 gene, and 4 pairs of primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., see Table 1 for details.

[0035] Table 1: RPA primers designed for the conserved region of the human bocavirus NP1 gene

[0036]

[0037] The primers in Table 1 were then screened by PCR amplification reaction.

[0038] The linearized plasmid frozen in Example 1 was used as a template for PCR amplification. The amplification system and conditions are shown in Table 2.

[0039] Table 2: PCR amplification conditions for human bocavirus NP1 gene with different primer pairs

[0040]

[0041]

[0042] Perform 2% agarose gel electrophoresis on the amplified product, load 6 μL of sample, 110V, 40min. The electrophoresis result is as follows figure 2 As shown, the brightness of the band amplified by...

Embodiment 3

[0055] Embodiment 3: specificity experiment

[0056] Using the same target sequence preparation method as in Example 1, adenovirus Hexon gene, human metapneumovirus N gene, and syncytial virus N gene were synthesized as templates respectively, and RPA amplification was performed using the preferred primer P3 in Example 2, For specific operations, refer to the instructions of the RPA amplification kit (RAA Nucleic Acid Amplification Kit, purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd., product number B00000), react at 39°C for 30 minutes, and detect by gel electrophoresis. Experimental results such as image 3 As shown, only human bocavirus is positive, and the others are all negative, indicating that the RPA detection system of the present invention has good specificity.

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Abstract

The invention discloses a kit and a method for rapidly detecting a human bocavirus based on isothermal amplification. A nucleotide sequence of a conserved gene NP1 protein of the human bocavirus is compared through multiple sequences, a DNA fragment with the size of 270bp is determined as a targeted analysis sequence, the sequence is located between the 10th site and the 279th site of the NP1 gene, and a specific RPA primer is designed based on the sequence. Further, a recombinase polymerase isothermal amplification method suitable for rapidly and accurately detecting the human bocavirus is optimized and established. The detection limit of the method reaches 252ag which is higher than that of a traditional PCR detection technology, and the kit has the advantages of being high in sensitivity, high in specificity, easy to operate, rapid and the like and is suitable for clinical rapid detection of the human bocavirus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a kit for rapidly detecting human bocavirus based on isothermal amplification and a method thereof. Background technique [0002] Human bocavirus (HBoV) is a parvovirus that has been shown to be a common cause of respiratory infections and gastroenteritis in children. HBoV is transmitted through the air and has a high infection rate. Symptoms are similar to those of the common cold. It is easy to cause pneumonia, bronchitis and bronchopneumonia in infants and young children. It is difficult to distinguish them from the common cold. The status and infection status are critical to the treatment of the disease. [0003] For the detection of human bocavirus, there are many methods, such as PCR amplification (droplet digital PCR, fluorescent quantitative PCR), which take a long time and need to rely on thermal cyclers with accurate temperature control, as well as expensive R...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2565/125
Inventor 徐荣李永东
Owner NINGBO MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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