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. 2013 Aug 1;8(8):e69941.
doi: 10.1371/journal.pone.0069941. Print 2013.

Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device

Yiyue Ge  1 , Bin Wu, Xian Qi, Kangchen Zhao, Xiling Guo, Yefei Zhu, Yuhua Qi, Zhiyang Shi, Minghao Zhou, Hua Wang, Lunbiao Cui
Affiliations

Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device

Yiyue Ge et al. PLoS One. .

Abstract

A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic showing location of RT-LAMP primer binding sites within HA and NA genes.
(A) HA RT-LAMP primer binding sites. Assay spans region from nucleotide 309–504 with reference to the HA gene sequence of the H7N9 virus strain A/Nanjing/1/2013 (H7N9). (B) NA RT-LAMP primer binding sites. Assay spans region from nucleotide 1097–1295 with reference to the NA gene sequence of the H7N9 virus strain A/Nanjing/1/2013 (H7N9).
Figure 2
Figure 2. Comparison of real-time turbidity measurement and LFD for the detection of RT-LAMP products.
HA (A and C) and NA (B and D) RT-LAMP on 10-fold serial dilutions of H7N9 virus RNA. (A and B) detection using the real-time turbidimetry device. DW: distilled water used as non-RNA negative control. (C and D) detection using LFD. Loop primers were tagged with Biotin and FITC.
Figure 3
Figure 3. Specificity test results of RT-LAMP-LFD for H7N9 virus detection.
The specificities of HA (A) and NA (B) RT-LAMP-LFD assays were determined by analyzing the RNA extracts from various control viruses and the reference virus. N: No template control; 1: human seasonal influenza A H1N1 virus; 2: human seasonal influenza A H3N2 virus; 3: human influenza B virus; 4: 2009 swine-origin influenza virus A H1N1 virus; 5: avian influenza A H5N1 virus; 6: avian influenza A H9N1 virus; 7: parainfluenza virus types 1; 8: parainfluenza virus types 2; 9: parainfluenza virus types 3; 10: parainfluenza virus types 4; 11: human coronavirus 229E; 12: human coronavirus OC43; 13: human coronavirus HKU1; 14: human coronavirus NL63; 15: respiratory syncytial viruses types A; 16: respiratory syncytial viruses types B; 17: H7N9 virus.
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References

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