靶向山羊痘病毒GPCR基因SYBR Green荧光定量PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.10.047

Abstract

Abstract:

Based on the genomic sequence of the goat pox virus (GTPV), we have established a fluorescence quantitative PCR method targeting the G-protein-coupled chemokine receptor (GPCR) gene. Six pairs of specific qPCR primers were designed based on the whole genomic sequence of GTPV. The specificity and sensitivity of these six qPCR primers were screened and detected. A pair of qPCR primers with high specificity and sensitivity was finally screened and obtained. According to the GPCR gene sequence of the GTPV AV41 vaccine strain, we designed an ordinary PCR primer pair to amplify the GPCR gene to construct the eukaryotic expression vector, which was used to establish the standard curve. Results showed that A pair of specific qPCR primers targeting the GPCR gene was obtained. The eukaryotic expression plasmid named pCAGGS-GPCR was constructed. The standard curve of y=-3.5289x+49.07 was established, whose linear correlation coefficient is R²=0.997 2 and amplification efficiency is 92.7%. The results of the replication experiment suggested that the lowest detective limitation of this primer was 2.0 copies·μL^-1, and the coefficient variation of the reproducibility results within and between batches was 2%, indicating the advantages of reasonable specificity, repeatability, and sensitivity. We have successfully established a specific fluorescence quantitative PCR method targeting the GPCR gene of GTPV, which provides adequate detecting support for the veterinary clinical diagnosis and the prevention and control of goat poxvirus.

Key words: goat pox virus, GPCR, fluorescence quantitative PCR

CLC Number:

S852.654

Hongqiang ZHANG, Shanhui REN, Wei YAO, Zhenli GONG, Xue YANG, Chunling MA, Ting YOU, Yuzhe ZHANG, Minyi LIU, Wenjie QIAN, Liuyang LI, Zhipeng YU, Yuefeng SUN, Haotai CHEN, Jiangfeng FAN. The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4779-4784.

Figures/Tables 5

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References 14

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引物名称Prime name		序列(5′→3′) Sequence
引物2(^#2) Primer 2	上游引物Forword primer	ACCAACACTACTTGTGCTATGA
	下游引物Reverse primer	GTATGGCATTAAGATTTGTCAACCT

引物名称 Primer		序列(5′→3′) Primer sequence	片段大小/bp Product size
引物7(PCR) Primer 7	上游引物 Forword primer	CATCATTTTGGCAAAGAATTCGCCACCATG- AATTATACGCTTAGCACAGTTAGTAGCAG	1 125
	下游引物 Reverse primer	GCTCGAGCATGCCCGGGTACCTCATTTATCGTCATCGTCTTTGT- AGTCGCTTCCTCCTCCTCCGATGCTAATACTACCAACACTACTTG	1 125

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The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus

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