Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification

doi:10.1186/s13568-018-0591-6

. 2018 Apr 18;8(1):60.

doi: 10.1186/s13568-018-0591-6.

Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification

Xu-Guang Guo^{1

2}, Yong-Zhuo Zhou³, Qin Li⁴, Wei Wang^{5

6}, Jin-Zhou Wen⁷, Lei Zheng¹, Qian Wang⁸

Affiliations

¹ Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong Province, China.
² Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, Guangdong Province, China.
³ Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510150, Guangdong Province, China.
⁴ Section of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Yale School of Medicine, Yale University, New Haven, CT, 06520-8057, USA.
⁵ Department of Laboratory Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen, 518035, Guangdong, China.
⁶ Department of Laboratory Medicine, Shenzhen Second People's Hospital, Shenzhen, 518035, Guangdong, China.
⁷ Center For Disease Control and Prevention of Chaozhou, Guangzhou, 521000, Guangdong Province, China.
⁸ Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong Province, China. wangqian@smu.edu.cn.

PMID: 29671176
PMCID: PMC5906417
DOI: 10.1186/s13568-018-0591-6

Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification

Xu-Guang Guo et al. AMB Express. 2018.

. 2018 Apr 18;8(1):60.

doi: 10.1186/s13568-018-0591-6.

Authors

Xu-Guang Guo^{1

2}, Yong-Zhuo Zhou³, Qin Li⁴, Wei Wang^{5

6}, Jin-Zhou Wen⁷, Lei Zheng¹, Qian Wang⁸

Affiliations

¹ Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong Province, China.
² Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, Guangdong Province, China.
³ Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510150, Guangdong Province, China.
⁴ Section of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Yale School of Medicine, Yale University, New Haven, CT, 06520-8057, USA.
⁵ Department of Laboratory Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen, 518035, Guangdong, China.
⁶ Department of Laboratory Medicine, Shenzhen Second People's Hospital, Shenzhen, 518035, Guangdong, China.
⁷ Center For Disease Control and Prevention of Chaozhou, Guangzhou, 521000, Guangdong Province, China.
⁸ Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong Province, China. wangqian@smu.edu.cn.

PMID: 29671176
PMCID: PMC5906417
DOI: 10.1186/s13568-018-0591-6

Abstract

To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.

Keywords: LAMP; Loop-mediated isothermal amplification; NS5 gene; Real-time fluorescence reverse transcription; Zika virus.

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Figures

**Fig. 1**
The primer of the LAMP screening test in this study: a real-amp without loop primers (NS5-1); b real-amp without loop primers (NS5-2); c real-amp without loop primers (NS5-3); d real-amp without loop primers (NS5-4)

**Fig. 2**
The LAMP melting curve in this study: a real-amp without loop primers (NS5-1); b real-amp without loop primers (NS5-2); c real-amp without loop primers (NS5-3); d real-amp without loop primers (NS5-4)

**Fig. 3**
The primer of LAMP screening test in this study. a Real-amp with loop primers (NS-1); b real-amp with loop primers (NS5-2); c real-amp with loop primers (NS5-3); d real-amp with loop primers (NS5-4)

**Fig. 4**
The LAMP melting curve in this study. a Real-amp with loop primers (NS5-1); b real-amp with loop primers (NS5-2); c real-amp with loop primers (NS5-3); d real-amp with loop primers (NS5-4)

**Fig. 5**
Sensitivity of LAMP for the amplification of Zika virus

**Fig. 6**
Specificity of LAMP for the amplification of Zika virus

**Fig. 7**
Repeatability of LAMP for the amplification of Zika virus

See this image and copyright information in PMC

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References

1. Abdul-Ghani R, Al-Mekhlafi AM, Karanis P. Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test? Acta Trop. 2012;122:233–240. doi: 10.1016/j.actatropica.2012.02.004. - DOI - PubMed
1. Ahmad F, Hashsham SA. Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review. Anal Chim Acta. 2012;733:1–15. doi: 10.1016/j.aca.2012.04.031. - DOI - PubMed
1. Asiello PJ, Baeumner AJ. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip. 2011;11:1420–1430. doi: 10.1039/c0lc00666a. - DOI - PubMed
1. Beck ET, Henrickson KJ. Molecular diagnosis of respiratory viruses. Future Microbiol. 2010;5:901–916. doi: 10.2217/fmb.10.48. - DOI - PubMed
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[1] Abdul-Ghani R, Al-Mekhlafi AM, Karanis P. Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test? Acta Trop. 2012;122:233–240. doi: 10.1016/j.actatropica.2012.02.004. - DOI - PubMed

[2] Abdul-Ghani R, Al-Mekhlafi AM, Karanis P. Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test? Acta Trop. 2012;122:233–240. doi: 10.1016/j.actatropica.2012.02.004. - DOI - PubMed

[3] Ahmad F, Hashsham SA. Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review. Anal Chim Acta. 2012;733:1–15. doi: 10.1016/j.aca.2012.04.031. - DOI - PubMed

[4] Ahmad F, Hashsham SA. Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review. Anal Chim Acta. 2012;733:1–15. doi: 10.1016/j.aca.2012.04.031. - DOI - PubMed

[5] Asiello PJ, Baeumner AJ. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip. 2011;11:1420–1430. doi: 10.1039/c0lc00666a. - DOI - PubMed

[6] Asiello PJ, Baeumner AJ. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip. 2011;11:1420–1430. doi: 10.1039/c0lc00666a. - DOI - PubMed

[7] Beck ET, Henrickson KJ. Molecular diagnosis of respiratory viruses. Future Microbiol. 2010;5:901–916. doi: 10.2217/fmb.10.48. - DOI - PubMed

[8] Beck ET, Henrickson KJ. Molecular diagnosis of respiratory viruses. Future Microbiol. 2010;5:901–916. doi: 10.2217/fmb.10.48. - DOI - PubMed

[9] Chen HL, Tang RB. Why Zika virus infection has become a public health concern? J Chin Med Assoc. 2016;79:174–178. doi: 10.1016/j.jcma.2016.03.001. - DOI - PubMed

[10] Chen HL, Tang RB. Why Zika virus infection has become a public health concern? J Chin Med Assoc. 2016;79:174–178. doi: 10.1016/j.jcma.2016.03.001. - DOI - PubMed

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Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification

Affiliations

Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification

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