SYBR Green实时荧光PCR快速鉴定枣实蝇技术

doi:10.11707/j.1001-7488.20140409

Abstract

Abstract:

The technique of a real-time PCR with SYBR Green I dye was used in this study. A SYBR Green real-time PCR was developed to rapidly obtained identify Carpomya vesuviana BIOER FQD-48A sequence detection system.A C.vesuviana specific PCR primers set was designed based on mtDNA COⅠ gene of C.vesuviana. Five Bactrocera fruit flies, B.dorsalis,B. cucurbitae,B. tau, B. correcta and B. zonata, were used to determine the specificity of the primers set CarF/CarR. A series of genomic DNA dilutions of C.vesuviana, 40, 20,10,1,0.1,0.01,0.001 ng·μL^-1, were used to detect the sensitivity of SYBR Green PCR. The results showed that the detection limit of SS-PCR was less than 0. 01 ng·μL^-1.The template DNA concentration was one of the sources of variability in cycle threshold values (CT) and the optimum DNA concentration was between 1 ng·μL^-1 and 20 ng·μL^-1 in SYBR Green PCR. The template DNA isolated from the larva, pupa and adult specimens of C. vesuviana respectively were used to detect the reliability of SYBR Green PCR. Melting curve analysis (MCA) and agarose gel electrophoresis (AGE) was then used to confirm the specificity reliability of PCR products, respectively. The similar amplification plots were obtained in three different stages of C.vesuviana.The average melting temperature (Tm) of the PCR product from B. latifrons was 75.3 ℃±0.1 ℃, and a 205 bp length fragment target was amplified.Except for the adult of fruit flies,the molecular techniques established in this study can also rapidly identify the larva and pupa.

Key words: Carpomya vesuviana, SYBR Green, real-time PCR, melting curve, rapid identification

CLC Number:

Q965

Cheng Xiaotian, Adil Sattar, Zhang Wei, Li Xinquan, Mayila. Rapid Identification of Carpomya vesuviana by Real-Time PCR with SYBR Green Chemical[J]. Scientia Silvae Sinicae, 2014, 50(4): 60-65.

References

阿地力·沙塔尔, 何善勇, 田呈明, 等.2008.枣实蝇在吐鲁番地区的发生及蛹的分布规律.植物检疫, 22(5): 295-297.
(Adili S, He S Y, Tian C M, et al. 2008. Occurrence of Carpomya vesuviana and distribution of pupas in Tulufan area. Plant Quarantine, 22(5): 295-297. [in Chinese] )
刘建越, 邓欣, 康林, 等. 2006.SYBR-Green实时荧光PCR检测转基因番木瓜.湖南农业大学学报:自然科学版, 32(4): 371-374.
(Liu J Y, Deng X, Kang L, et al. 2006. Real-time fluorescent SYBR-Green PCR detect the genetically modified papaya. Journal of Hunan Agricultural University:Natural Sciences, 32(4): 371-374. [in Chinese] )
吴佳教, 胡学难, 赵菊鹏, 等. 2005.9种检疫性实蝇PCR-RFLP快速鉴定研究.植物检疫, 19(1): 2-6.
(Wu J J, Hu X N, Zhao J P, et al. 2005. Rapid identification among 9 species of quarantine fruit flies(Diptera:Tephritidae)by PCR-RFLP. Plant Quarantine, 19(1): 2-6. [in Chinese] )
易建平, 刘素萍, 印丽萍, 等. 2005.TaqMan-MGB探针在小麦印度腥黑穗病菌和黑麦草腥黑穗病菌鉴定上的应用.植物检疫, 19(1): 15-19.
(Yi J P, Liu S P, Yin L P, et al. 2005. Use of TaqMan- MGB probe for identification of Tilletia indica and T.walkeri. Plant Quarantine, 19(1): 15-19. [in Chinese] )
余道坚.2005.检疫性实蝇分子生物学快速鉴定技术的研究.上海: 中国科学院上海生命科学研究院博士学位论文.
(Yu D J. 2005. Study on rapid identification of quarantine fruit fly (diptera: tephritidae) based on molecular techniques. Shanghai: PhD thesis of Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences The Chinese Academy of Sciences. [in Chinese] )
余道坚, 章桂明, 陈志粦, 等. 2006.SYBR Green实时荧光PCR快速鉴定辣椒实蝇.植物检疫, 20(1): 10-14.
(Yu D J, Zhang G M, Chen Z L, et al. 2006. Rapid identification of Bactrocera latifrons by real-time PCR using SYBR Green chemistry. Plant Quapantine, 20(1): 10-14. [in Chinese] )
张润志, 汪兴鉴, 阿地力·沙塔尔.2007.检疫性害虫枣实蝇的鉴定与入侵威胁.昆虫知识, 44(6): 928-930.
(Zhang R Z, Wang X J, Adili S. 2007. Identification and precaution of the ber fruit fly, Carpomya vesuviana, a quarantine pest insect in China. Chinese Journal of applied entomology, 44(6): 928-930. [in Chinese] )
Giolio S, Monis P T, Saint C P. 2003.Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR.Nucleic Acids Research, 31 (22): 136-137.
Giulietti A, Overbergh L, Valckx D, et al.. 2001.An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.Methods, 25(4): 386-401.
Muraji M, Nakahara S. 2002.Discrimination among pest species of Bactrocera (Diptera, Tephritidae) based on PCR-RFLP of the mitochondrial DNA.Applied Entomology and Zoology, 37(3): 437-446.
Naeole C K M, Haymer D S.2003.Use of oligonucleotide arrays for molecular taxonomic studies of closely related species in the oriental fruit fly (Bactrocera dorsalis) complex. Molecular Ecology Notes, 3(4): 662-66.
Nakahara S, Kato H, Kaneda M, et al.2002. Identification of the Bactrocera dorsalis Complex (Diptera: Tephritidae) by PCR-RFLP Analysis. Ⅲ. Discrimination between B. philippinensis and B. occipitalis.Res Bull Pl Prot Japan, 38(4): 73-80.
Ponchel F, Toomes C, Bransfield K, et al.2003. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotech, 3: 18.
Tanriverdi S, Tanyeli A, Baslamish F, et al. 2002.Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. Journal of Clinical Microbiology, 40(9): 3237-3244.

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Rapid Identification of Carpomya vesuviana by Real-Time PCR with SYBR Green Chemical

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