Typing detection LAMP primer combination with effect of differentiating porcine parvovirus type 1-7, detection method and kit

A parvovirus and primer combination technology, applied in the biological field, can solve the problems of aerosol pollution, false positive results, inability to distinguish between specific amplification and non-specific amplification, etc.

Active Publication Date: 2019-11-12
北京市动物疫病预防控制中心 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Moreover, most of the currently established LAMP reaction methods require agarose gel electrophoresis or need to add a chromogenic reagent after opening the cover, which is likely to cause laboratory contamination or lead to false positive results.
In addition, most of the chromogenic methods used by the LAMP method for direct observation are to open the cover after the reaction and add fluorescent dyes for color reaction, and observe whether there is color development to interpret the test results, which will cause aerosol pollution and cannot distinguish specific amplification. and non-specific amplification, thus increasing the probability of false positive diagnostic results; and for weak positive reactions, it is likely to be misjudged as negative by human naked eyes

Method used

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  • Typing detection LAMP primer combination with effect of differentiating porcine parvovirus type 1-7, detection method and kit
  • Typing detection LAMP primer combination with effect of differentiating porcine parvovirus type 1-7, detection method and kit
  • Typing detection LAMP primer combination with effect of differentiating porcine parvovirus type 1-7, detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1, the screening preparation of primer combination

[0115] 1. Screening of primer combinations

[0116] 1. The primer sequences used were synthesized by Sangon Company, and several sets of primers were designed for porcine parvovirus NS1 or VP2 genes. The sequences of each set of primers are shown in Table 1.

[0117] Table 1 Primer Set Sequence

[0118]

[0119]

[0120]

[0121]

[0122] In the above primer combinations, each single-stranded DNA is packaged independently.

[0123] In the above primer combinations, the molar ratios of primer F3, primer B3, primer FIP, primer BIP, primer LF and primer LB in each primer set are all 0.3:0.3:2.4:2.4:1:1.

[0124] 2. Using the porcine parvovirus 1-7 detection gene plasmid DNA as a template, respectively use the primer set prepared in step 1 to perform loop-mediated isothermal amplification detection on the template.

[0125] Porcine parvovirus type 1 detection gene plasmid: Insert Genebank numbers...

Embodiment 2

[0144] Embodiment 2, preliminary screening of sensitivity

[0145] Sample to be tested: the plasmid of porcine parvovirus 1-7 prepared in Example 1.

[0146] 1. Extract the plasmid DNA of the sample to be tested, and perform gradient dilution with sterile water to obtain each dilution.

[0147] 2. Using the dilution obtained in step 1 as a template, the primer combination prepared in Example 1 was used to perform loop-mediated isothermal amplification.

[0148] Reaction system (20 μL): 10 μL reaction solution (product of Boao Biological Group Co., Ltd., catalog number: CP.440020), 2.96 μL primer mixture, 2 μL template diluent (1 μL of the genome copy number contained in the diluent is 10 4 and 10 3 ), replenish water to 20 μL. The primer mixture is the mixture of each primer in the primer combination. In the reaction system, 0.12 μL each of 0.3 mM outer primers F3 and B3; 0.96 μL each of 2.4 mM inner primers FIP and BIP; 0.4 μL each of 1 mM loop primers LF and LB.

[0149...

Embodiment 3

[0163] Embodiment 3, sensitivity double screening

[0164] Sample to be tested: the plasmid of porcine parvovirus 1-7 prepared in Example 1.

[0165] 1. Extract the plasmid DNA of the sample to be tested, and perform gradient dilution with sterile water to obtain each dilution.

[0166] 2. Using the dilution obtained in step 1 as a template, the primer combination prepared in Examples 1 and 2 was used to perform loop-mediated isothermal amplification.

[0167] Reaction system (20 μL): 10 μL reaction solution (product of Boao Biological Group Co., Ltd., catalog number: CP.440020), 2.96 μL primer mixture, 2 μL template diluent (1 μL of the genome copy number contained in the diluent is 10 3 、10 2 or 10 1 ), replenish water to 20 μL. The primer mixture is the mixture of each primer in the primer combination. In the reaction system, 0.12 μL each of 0.3 mM outer primers F3 and B3; 0.96 μL each of 2.4 mM inner primers FIP and BIP; 0.4 μL each of 1 mM loop primers LF and LB.

...

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Abstract

The invention belongs to the technical field of biology and discloses a typing detection LAMP primer combination with an effect of differentiating porcine parvovirus type 1-7, a detection method and akit. Multiple sets of LAMP primers are designed respectively by utilizing conserved segments of the NS1 gene and NP2 gene of a porcine parvovirus and are screened to obtain the typing detection primer combination which has good specificity and high sensitivity and can be used for differentiating the porcine parvovirus type 1-7. The primer combination can be used for respectively detecting porcineparvovirus type 1-7 and does not have a cross reaction with other porcine viruses. A fluorescent dye is added into a LAMP reaction system, the porcine parvovirus type 1-7 can be detected by a one-step method, whole-process real-time monitoring can be implemented by combining fluorescent quantitation, a result can be obtained within 1 hour, and the detection sensitivity can be 100 copies / [mu]L. The detection method has the advantages of convenience, quickness, sensitiveness and specificity and can avoid an error caused by human determination and environmental pollution caused by cap opening.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer combination, a detection method and a kit for LAMP capable of distinguishing porcine parvovirus 1-7 types for typing and detection, and is used for detecting and distinguishing porcine parvovirus 1-7 types. Background technique [0002] Porcine parvovirus (porcine parvovirus) is one of the main pathogens that cause reproductive disorders in pregnant sows, and is characterized by infected sows, especially primiparous sows, showing abortion, stillbirth, mummified fetuses and deformed fetuses, or The small number of litters can sometimes lead to infertility in male and female pigs. Porcine parvovirus is highly infectious. Once the virus is introduced into susceptible healthy pigs, it can cause almost 100% infection within 3 months; the pigs in the infected group remain positive for a long time. Porcine parvovirus mostly occurs in spring and summer or sow farrowing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 张岩马银平陈燕旌邢婉丽程京
Owner 北京市动物疫病预防控制中心
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