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Primer for detecting FHV-1, nucleic acid capture gold-labeled test strip, reagent kit and application

A FHV-1, nucleic acid capture technology, applied in the field of molecular biology, can solve the problems of high operator requirements, large instrument cost investment, low sensitivity, etc., and achieve improved detection sensitivity, high amplification efficiency and specificity, and high sensitivity. Effect

Inactive Publication Date: 2019-02-15
苏州蝌蚪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Common methods for detecting FHV-1 at present include: enzyme-linked immunoassay, but this method has low sensitivity and high false positives, which is easy to cause false detection; fluorescent quantitative PCR method, this method has high sensitivity, but the cost of equipment is large, and it is difficult for operation. Personnel requirements are higher
In addition, there are dot hybridization, southern hybridization, etc., but these methods have high false positives, cumbersome operations, and take a long time

Method used

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  • Primer for detecting FHV-1, nucleic acid capture gold-labeled test strip, reagent kit and application
  • Primer for detecting FHV-1, nucleic acid capture gold-labeled test strip, reagent kit and application
  • Primer for detecting FHV-1, nucleic acid capture gold-labeled test strip, reagent kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1 is used for carrying out the primer design of PCR to FHV-1

[0105] Referring to the FHV-1 genome sequence included in GenBank, select the conserved region of the gC gene of FHV-1, and use ABIPrimerExpress 3.0 PCR primer design software to design and synthesize primers.

[0106] The typical primer pair sequences are as follows:

[0107]

[0108]

[0109] like figure 1 As shown, the four pairs of primers designed for FHV-1 virus were used for PCR amplification experiments, and the electrophoresis results showed that the primer amplification efficiency of FHV / gC210 was the highest, so this experiment used FHV / gC210-F and FHV / gC210- R is used as the detection sequence of FHV-1 virus, and its sequences are shown in SEQ ID NO.15 and SEQ ID NO.16 respectively.

[0110] in figure 2 The amplified fragment band numbered 1 is the PCR amplification product of the primer pair FHV / gC244; figure 2 The amplified fragment band numbered 2 is the PCR amplificatio...

Embodiment 2

[0111] Embodiment 2 PCR experiment

[0112] 1. Magnetic bead method for nucleic acid extraction

[0113] 1.1 Sample processing

[0114] Take pet feces or nasal secretion samples in 1mL normal saline or 1×PBS (0.01M, pH7.2) to prepare homogenate. Centrifuge at 2000rpm for 10min, and transfer the supernatant to a new centrifuge tube.

[0115] 1.2 Nucleic acid preparation

[0116] 1.2.1 After mixing the sample liquid evenly under sterile conditions, take 300 μL of the sample into a 1.5 mL clean centrifuge tube, add 500 μL of lysate and 10 μL of proteinase K, and bathe in water at 56°C for 30 minutes.

[0117] 1.2.2 The magnetic beads must be incubated at 37°C for 10 minutes before use, and shake gently by hand to make them appear in a suspended state.

[0118] 1.2.3 Add 15 μL of heated and mixed magnetic beads to the sample that has been lysed in a water bath, then add 250 μL of binding solution, and vortex to mix.

[0119] 1.2.4 Rotate the centrifuge tube in a mixer for 10 ...

Embodiment 3

[0137] Embodiment 3, colloidal gold standard test paper strip making

[0138] 3.1 Goldmaking

[0139] 3.1.1 Add 1ml of chloroauric acid (5g / L) to a clean Erlenmeyer flask equipped with a magnetic stirring bar, dilute it to 50ml with ultrapure water, and seal it with plastic wrap.

[0140] 3.1.2 Put the Erlenmeyer flask on the magnetic stirrer, turn on the stirrer, adjust the position of the Erlenmeyer flask so that the stirring bar is in the middle of the Erlenmeyer flask, and slowly increase the stirring speed to 1300±20r / min to ensure magnetic stirring After the child is stirring stably in the center of the Erlenmeyer flask, turn off the stirrer, stop stirring, adjust the heating knob to the maximum, and start heating.

[0141] 3.1.3 When the liquid in the bottle starts to boil, turn on the stirrer and quickly add 1000 μL of trisodium citrate solution (1%) into the bottle at the same time.

[0142] 3.1.4 Continue heating and stirring. After the color of the liquid graduall...

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Abstract

The invention discloses a primer for detecting FHV-1, a nucleic acid capture gold-labeled test strip, a reagent kit and an application. A primer pair for detecting FHV-1 comprises a first primer and asecond primer with sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8 respectively. The gold-labeled test strip comprises a lining plate, wherein a sample pad, a gold-labeled combining pad combined with an FITC gold-labeled antibody, a coating membrane and a water absorption pad are sequentially arranged on the upper end surface of the lining plate in a set direction; an invisible detection area combined with a digoxin antibody and an invisible control area combined with a sheep anti-rat secondary antibody are arranged on the coating membrane. The reagent kit comprises the primer pair and the nucleic acid capture gold-labeled test strip. A detection method of FHV-1 is high in specificity and sensitivity, accurate and quick, and has wide application prospects.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a PCR amplification combined with nucleic acid molecule capture technology, primers for detecting FHV-1 virus, nucleic acid capture gold standard test strips, corresponding kits and detection applications thereof. Background technique [0002] Feline herpes virus (FHV-1) is a large virus (100-130nm in diameter), has an envelope and double-stranded DNA, proliferates in the nucleus, and forms nuclear inclusion bodies. [0003] Feline viral rhinotracheitis is an upper respiratory infectious disease caused by feline herpes virus. It is transmitted between cats and cats, and the mortality rate is extremely high. The early clinical symptoms include: depression, sneezing and coughing, followed by Photophobia, conjunctivitis, and serous eye and nasal discharge, body temperature and rapid rise, paroxysmal sneezing, deep tracheal cough, tongue and maxillary ulcers and other sympt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6804C12N15/11C12R1/93
CPCC12Q1/6804C12Q1/705C12Q2531/113C12Q2565/625C12Q2563/131
Inventor 薛峰陈伟苏静周莉质陆寿祥吴城霖
Owner 苏州蝌蚪生物技术有限公司
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