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A lamp kit for rapid color development and one-step detection of porcine parvovirus

A technology of parvoviruses and kits, applied in recombinant DNA technology, biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve problems such as cumbersome operations, increased false positive rate, and decreased amount of amplified products, and achieve simplification Operating procedure, good specificity, effect of improving amplification efficiency

Active Publication Date: 2019-07-26
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After adding SYBRGreen dye, the positive reaction tube can form a brown color, which can be recognized by naked eyes, but the brown color is not as easy to observe as the bright green formed by adding calcein premixed dye
Calcein-containing fluorescent dyes can inhibit Bst polymerase to a certain extent, so adding these dyes directly to the reaction tube will lead to a decrease in the amount of amplification products, and it is difficult to form typical ladder-shaped bands on agarose gel electrophoresis , it is also difficult to observe the change in the fluorescence of the reaction tube
The two-step method of adding fluorescent dyes to observe and judge after the reaction is not only cumbersome to operate, but also the main problem is that when the reaction tube cover is opened, the amplification product is released into the air, forming a large amount of aerosol, and the reaction is repeated. When LAMP is detected, the false positive rate will greatly increase

Method used

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  • A lamp kit for rapid color development and one-step detection of porcine parvovirus
  • A lamp kit for rapid color development and one-step detection of porcine parvovirus
  • A lamp kit for rapid color development and one-step detection of porcine parvovirus

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 Preparation of the kit of the present invention

[0041] 1. Extraction of sample DNA

[0042]The MiniBEST Viral RNA / DNAExtraction Kit produced by Takara Bao Bioengineering (Dalian) Co., Ltd. was used to extract nucleic acids from cytotoxicity, serum and ground tissue samples.

[0043] 2. Use different methods to determine the PPV in the sample

[0044] 1. Determination of PPV by conventional PCR reaction experiment

[0045] PPV conventional PCR amplification primer sequence

[0046] Table 1 PPV type 1 routine PCR amplification primer sequence

[0047]

[0048] P16-F and P17-R are used together as a primer pair for the identification of PPV porcine parvovirus by conventional PCR, targeting the 461-905bp fragment region of the capsid protein VP2 gene, and the estimated size of the amplified product is 445bp.

[0049] PCR amplification reaction system (20μl)

[0050] PrimeSTAR® GXL DNA Polymerase 0.4μL

[0051] dNTP Mixture 1.6μL

[0052] 5×PrimeSTAR...

Embodiment 2

[0081] Embodiment 2 Performance experiment of the kit of the present invention

[0082] 1. Sensitivity experiment of PPV one-step rapid chromogenic LAMP analysis

[0083] AV30 strain (CVCCAV30) has high homology with the classic strain NADL-2, so this experiment refers to the genome sequence of NADL-2 strain to calculate the copy number of extracted nucleic acid.

[0084] Copy number calculation formula:

[0085] (6.02×10 23 copy number / mole)×(concentration g / ml) / (MW g / mol)=copy number / ml

[0086] or (6.02×10 23 copy number / mole)×(concentration ng / μl)×10 -6 / (MW g / mol)=copy number / ml

[0087] [Average molecular weight (MW g / mol): dsDNA=(number of bases)×(660 Daltons / base); ssDNA=(number of bases)×(330 Daltons / base); ssRNA=(number of bases) base)×(340 Daltons / base)].

[0088] PPV belongs to single-stranded DNA, and the full genome length of NADL-2 reference strain is 5075bp, so its average molecular weight MW=330×5075=1674750 Daltons.

[0089] AV30 strain DNA copy numbe...

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Abstract

The invention provides a rapidly color developing LAMP kit for one-step detection of porcine parvoviruses. The kit comprises a detection system which is constituted by LAMP reaction liquid of six LAMP primers. The kit provided by the invention, under the condition of not prolonging an original reaction time, namely 1h, achieves one-step operation of directly adding a fluorescent dye to a reaction tube, so that an operating procedure is simplified and the fundamental problem that it fails to develop color when the fluorescent dye is directly added to the reaction tube due to inhibition of a premixed dye containing calcein on a Bst DNA amplification enzyme is solved, and meanwhile, the occurrence of pollution caused by an aerosol which is formed by a product formed by secondary uncovering after a reaction is effectively prevented. The kit provided by the invention, when adopted in detection, can achieve intuitive judgment directly by naked eyes; and the kit is good in specificity, sensitivity and stability.

Description

technical field [0001] The invention relates to a LAMP kit for detecting porcine parvovirus by rapid color development and one-step method. Background technique [0002] Porcine parvovirus (PPV) can cause reproductive disorders in sows and fattening pigs. It is one of the most common porcine reproductive disorder viruses that bring serious economic losses to pig farms. It is still widely prevalent in pig farms in my country. Since most pig farms in my country, especially large-scale farms, widely use PPV-type inactivated vaccines to vaccinate pigs, it is basically impossible to distinguish infection and immunity by serological methods such as ELISA. Therefore, the census or diagnosis of PPV infection mainly depends on various diagnostic methods based on the detection of nucleic acid. Common PPV nucleic acid diagnostic methods include conventional PCR, real-time quantitative PCR, and in situ hybridization. These methods are mainly used in various laboratories due to the limi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 张杰张永光刘永生丁耀忠陈豪泰常惠芸吕建亮林彤潘丽刘新生王永录
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI