Calcein visual RT-LAMP kit and application thereof

Abstract

The invention discloses a calcein visual RT-LAMP kit and application thereof. The kit is characterized by containing a primer group, calcein, manganese chloride, a novel duck reovirus RNA template, RNase-free water, a 10-time reaction buffer solution, dNTPs, glycine betaine, MgSO4, Bst DNA polymerase and AMV reverse transcriptase, wherein the primer group comprises an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP, a downstream inner primer BIP, a loop primer LoopF and a loop primer LoopB, and the sequences of the primers are presented by SEQ ID NO: 1-6. According to the calcein visual RT-LAMP kit, the problems that a detection method for detecting the novel duck reovirus is complicated in operation, low in sensitivity and long in consumed time and cannot meet rapid field or base-layer detection requirements in the prior art are solved.

Description

technical field

[0001] The invention relates to the field of biological detection, in particular to a calcein visualized RT-LAMP kit and its application. Background technique

[0002] New-type duck reovirus (NDRV) disease is a new disease characterized by irregular liver necrosis, hemorrhagic spots and myocardial and bursa hemorrhage. The main clinical symptoms of sick ducks are low energy, reduced feed intake or waste. Some ducks have white and loose stools, acute onset, and short course of disease (within 24 hours). Soft feet phenomenon. The virus is different from Muscovy duck reovirus in terms of pathogenicity, antigenicity and gene sequence (Huang Yu, 2009; Chen Zongyan et al., 2012; Yuan Yuanhua et al., 2013). Since 2005, the disease has been spreading from Fujian, Guangdong, Zhejiang and other regions to other regions, which has brought huge economic losses to the development of the poultry industry.

[0003] Currently, the commonly used detection methods for the n...

Examples

Embodiment 1

[0077] Example 1: Primer Design

[0078] Using the sigmaB gene sequence of the DRV-QY strain as a design template (full length 1104bp), several sets of primers were designed using the LAMP online primer design software Primer Explorer4, and the primers were screened by using Larsergene7.0PrimerSelect and Oligo7.0, and searched by Primer-BLAST The specificity of the designed primers should not only ensure that each set of primers has a better theoretical value when performing RT-LAMP amplification, but also ensure that each primer pair produces the least dimer and has good conservation and specificity. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the designed and screened primers were as follows:

[0079] Table 1 RT-LAMP calcein visualization primer (Primer1)

[0080]

[0081]

[0082] The positions of RT-LAMP calcein visualization primers in the gene are as follows:

[0083]

[0084] (Note: The four "CCGG" bases in b...

Embodiment 2

[0085] Example 2: Identification of primer specificity by enzyme digestion

[0086] The RT-LAMP product of Primer1 was digested with HpaⅡ enzyme, and then detected by 2% agarose gel electrophoresis, the results were as follows: figure 1 As shown, there is a restriction band near 100 bp in the range of 100 bp to 250 bp, which is consistent with the estimated size of the two bands (127 bp and 114 bp), indicating that the restriction specificity of the RT-LAMP product of Primer1 is good.

[0087] The application of the calcein visualization RT-LAMP kit of the new duck reovirus, including steps:

[0088] S1. Prepare RT-LAMP reaction system: add calcein, manganese chloride, magnesium sulfate, betaine, dNTPs, AMV enzyme, Bst polymerase, 10 times reaction buffer, upstream external primer F3, downstream external primer B3, upstream internal Primer FIP, downstream inner primer BIP, loop primer LoopF, loop primer LB and the sample to be tested;

[0089] S2. Reaction: react the reactio...

Embodiment 3

[0095] Example 3: Primer specificity verification

[0096] RT-LAMP specificity tests were performed on DRV, MDRV, ARV, DPV, DHAV, ND, H9+H5, H7, Duck Tambusu virus (DTMUV) with Primer1 primers, followed by fluorescence observation and 2% agarose gel Electrophoresis detection, the results are as follows figure 2 and image 3 As shown, it shows that the Primer1 primer has good specificity to DRV.

technical field

[0001] The invention relates to the field of biological detection, in particular to a calcein visualized RT-LAMP kit and its application. Background technique

[0002] New-type duck reovirus (NDRV) disease is a new disease characterized by irregular liver necrosis, hemorrhagic spots and myocardial and bursa hemorrhage. The main clinical symptoms of sick ducks are low energy, reduced feed intake or waste. Some ducks have white and loose stools, acute onset, and short course of disease (within 24 hours). Soft feet phenomenon. The virus is different from Muscovy duck reovirus in terms of pathogenicity, antigenicity and gene sequence (Huang Yu, 2009; Chen Zongyan et al., 2012; Yuan Yuanhua et al., 2013). Since 2005, the disease has been spreading from Fujian, Guangdong, Zhejiang and other regions to other regions, which has brought huge economic losses to the development of the poultry industry.

[0003] Currently, the commonly used detection methods for the n...

Examples

Embodiment 1

[0077] Example 1: Primer Design

[0078] Using the sigmaB gene sequence of the DRV-QY strain as a design template (full length 1104bp), several sets of primers were designed using the LAMP online primer design software Primer Explorer4, and the primers were screened by using Larsergene7.0PrimerSelect and Oligo7.0, and searched by Primer-BLAST The specificity of the designed primers should not only ensure that each set of primers has a better theoretical value when performing RT-LAMP amplification, but also ensure that each primer pair produces the least dimer and has good conservation and specificity. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the designed and screened primers were as follows:

[0079] Table 1 RT-LAMP calcein visualization primer (Primer1)

[0080]

[0081]

[0082] The positions of RT-LAMP calcein visualization primers in the gene are as follows:

[0083]

[0084] (Note: The four "CCGG" bases in b...

Embodiment 2

[0085] Example 2: Identification of primer specificity by enzyme digestion

[0086] The RT-LAMP product of Primer1 was digested with HpaⅡ enzyme, and then detected by 2% agarose gel electrophoresis, the results were as follows: figure 1 As shown, there is a restriction band near 100 bp in the range of 100 bp to 250 bp, which is consistent with the estimated size of the two bands (127 bp and 114 bp), indicating that the restriction specificity of the RT-LAMP product of Primer1 is good.

[0087] The application of the calcein visualization RT-LAMP kit of the new duck reovirus, including steps:

[0088] S1. Prepare RT-LAMP reaction system: add calcein, manganese chloride, magnesium sulfate, betaine, dNTPs, AMV enzyme, Bst polymerase, 10 times reaction buffer, upstream external primer F3, downstream external primer B3, upstream internal Primer FIP, downstream inner primer BIP, loop primer LoopF, loop primer LB and the sample to be tested;

[0089] S2. Reaction: react the reactio...

Embodiment 3

[0095] Example 3: Primer specificity verification

[0096] RT-LAMP specificity tests were performed on DRV, MDRV, ARV, DPV, DHAV, ND, H9+H5, H7, Duck Tambusu virus (DTMUV) with Primer1 primers, followed by fluorescence observation and 2% agarose gel Electrophoresis detection, the results are as follows figure 2 and image 3 As shown, it shows that the Primer1 primer has good specificity to DRV.

Images