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. 2011 Jul;175(1):133-6.
doi: 10.1016/j.jviromet.2011.04.024. Epub 2011 Apr 27.

Development of loop-mediated isothermal amplification assay for detection of human coronavirus-NL63

Krzysztof Pyrc  1 , Aleksandra Milewska, Jan Potempa
Affiliations

Development of loop-mediated isothermal amplification assay for detection of human coronavirus-NL63

Krzysztof Pyrc et al. J Virol Methods. 2011 Jul.

Abstract

Human coronavirus NL63 was identified in 2004 in the Netherlands. Due to the high prevalence and world-wide distribution of this pathogen, it is essential to develop a sensitive and specific detection assay suitable for use in a routine diagnostic laboratory. Techniques based on PCR or real-time PCR are laborious and expensive. Detailed analysis of the HCoV-NL63 genome permitted the identification of a conserved nucleic acid sequential motif, which was sufficient for the design of a loop-mediated isothermal amplification (LAMP) assay. Evaluation of the method showed that the test is specific to HCoV-NL63 and that it does not cross-react with other respiratory viruses. The detection limit was found to be 1 copy of RNA template per reaction in cell culture supernatants and clinical specimens.

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Figures

Fig. 1
Fig. 1
Sensitivity of the HCoV-NL63 LAMP assay. The assay was performed with serial dilutions of viral RNA (105, 104, 103, 102, 10, 5, and 1 copies per reaction). Mock: negative control from mock-infected LLC-MK2 cells.
Fig. 2
Fig. 2
Cross-reactivity of the HCoV-NL63 LAMP assay. 1: HCoV-NL63; 2: HCoV-229E; 3: HCoV-HKU1; 4: respiratory syncytial virus; 5: echovirus 9; 6: human metapneumovirus; 7: influenza A virus; 8: adenovirus type 1.
Fig. 3
Fig. 3
Detection of HCoV-NL63 in clinical specimens with the LAMP assay. 1: Cell culture supernatant; 2: bronchoalveolar lavage; 3: nose wash; 4: sputum; 5: human sera; Mock: negative control from mock-infected LLC-MK2 cells.
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