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J Virol Methods. 2014 Feb;196:204-11. doi: 10.1016/j.jviromet.2013.11.012. Epub 2013 Nov 28.

Extraction of total nucleic acid based on silica-coated magnetic particles for RT-qPCR detection of plant RNA virus/viroid.

Author information

1
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China; Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China; CAS Key Laboratory of Measurement and Standardization for Nanotechnology, National Center for Nanoscience and Technology, Beijing 100190, China.
2
Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China.
3
CAS Key Laboratory of Measurement and Standardization for Nanotechnology, National Center for Nanoscience and Technology, Beijing 100190, China.
4
Grirem Advanced Materials Co., Ltd., National Engineering Research Center for Rare Earth Materials, General Research Institute for Nonferrous Metals, Beijing 100088, China.
5
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China. Electronic address: xiaq@seu.edu.cn.

Abstract

In this study, a nucleic acid extraction method based on silica-coated magnetic particles (SMPs) and RT-qPCR assay was developed to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The amplification sequences of RT-qPCR were reversely transcribed in vitro as RNA standard templates. The standard curves covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. Extraction efficiency of the SMPs method was evaluated by recovering spiked ssRNAs from plant samples and compared to two commercial kits (TRIzol and RNeasy Plant mini kit). Results showed that the recovery rate of SMPs method was comparable to the commercial kits when spiked ssRNAs were extracted from lily leaves, whereas it was two or three times higher than commercial kits when spiked ssRNAs were extracted from grapevine leaves. SMPs method was also used to extract viral nucleic acid from15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples. SMPs method did not show statistically significant difference from other methods on detecting ArMV, but LSV. The SMPs method has the same level of virus load as the TRIzol, and its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by RT-qPCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. In conclusion, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, especially grapevine viroids, from lily-leaf or grapevine-leaf samples for RT-qPCR detection.

KEYWORDS:

Nucleic acid extraction; RT-qPCR; Silica-coated magnetic particles; Virus/viroid

PMID:
24291163
DOI:
10.1016/j.jviromet.2013.11.012
[Indexed for MEDLINE]

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