Development of Armored RNA Reference Material of Norovirus Based on Qbeta Bacteriophage

doi:10.13523/j.cb.20180105

China Biotechnology

2018, Vol. 38

Issue (1): 42-50 DOI: 10.13523/j.cb.20180105

Orginal Article

Development of Armored RNA Reference Material of Norovirus Based on Qbeta Bacteriophage

Qi ZHANG¹,Lin YAO²,Yan-hua JIANG²,Feng-ling LI²,Yuan ZHANG³,Dong-qin XU¹,Wen-jia ZHU²,Ying-ying GUO²,Lian-zhu WANG²(

),Yu-xiu ZHAI²

1 Shanghai Ocean University, Shanghai 201306,China
2 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality,Ministry of Agriculture, P. R. China, Laboratory of Quality & Safety Risk Assessment for Aquatic Products (Qingdao), Ministry of Agriculture, P. R. China, Qingdao 266071, China;
3 Zhangzidao Group Co., LTD, Dalian 116001, China;

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Abstract Objective:

To develop armored RNA reference material containing target RNA of norovirus based on Qbeta bacteriophage for norovirus nucleic acid detection.

Methods:

Synthesize DNA fragment named QINVGII containing maturase coding gene, capsid protein coding gene and packing site of Qbeta bacteriophage, cDNA corresponding detection target sequence of gene group II norovirus in ISO/T15216-2 2012. QINVGII fragment was cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid was identified by enzyme digestion and sequencing, and then expressed in E. coli BL21 (DE3) cells through isopropyl-β-thiogalactopyranoside (IPTG) induction. The expression product, VLPs was analyzed by SDS-PAGE and the electron microscope. The VLPs was centrifuged and purified by CsCl density gradient after digestion with DNase I and RNase A. The homogeneity and stability of the reference material were tested according to the GB/T15000.3—2008 [directives for the work of reference materials (3) - reference material -general and statistical principles for certification].

Results:

SDS-PAGE analysis showed that the molecular mass of the expressed protein product was about 14.1kDa, which was consistent with the prediction. The 25nm VLPs could be observed under electron microscope. The VLPs samples were valued as (1.06±0.06)×10⁷ copies/μl and behaved well in the homogeneity test, F=2.24<F_0.05(9,20). The stability test indicated that the sample was stable at 37℃ for 12 days, at room temperature (20~25℃) for 24 days, at 4℃ for at least 120 days, -20℃ for at least 150 days, at -80℃ for at least 300 days with no significant decrease.

Conclusion:

The armored RNA based on Qbeta bacteriophage prepared, which had good uniformity, stability and high copy number, could be a good reference material candidate for the norovirus RNA detection.

Key words： Norovirus Armored RNA Qbeta bacteriophage Reference material Real-time RT-PCR

Received: 31 May 2017 Published: 31 January 2018

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	Qi ZHANG
	Lin YAO
	Yan-hua JIANG
	Feng-ling LI
	Yuan ZHANG
	Dong-qin XU
	Wen-jia ZHU
	Ying-ying GUO
	Lian-zhu WANG
	Yu-xiu ZHAI

Cite this article:

Qi ZHANG,Lin YAO,Yan-hua JIANG,Feng-ling LI,Yuan ZHANG,Dong-qin XU,Wen-jia ZHU,Ying-ying GUO,Lian-zhu WANG,Yu-xiu ZHAI. Development of Armored RNA Reference Material of Norovirus Based on Qbeta Bacteriophage. China Biotechnology, 2018, 38(1): 42-50.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180105 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I1/42

Table 1 Information of primers and probe

Fig.1 PCR amplification of QINVGII

Fig.2 Identification of pET-QINVGII by restriction analysis

Fig.3 SDS-PAGE analysis of VLPs expression

Fig.4 Electron microscopy of VLPs 400 000×

Fig.5 Agarose gel analysis of residual plasmid detection in VLPs

Table 2 Valuation of VLPs

Table 3 Variance analysis of homogeneity test

Table 4 Trend test for stability of Norovirus VLPs


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