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. 2020 Dec 11;20(1):947.
doi: 10.1186/s12879-020-05585-4.

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

Boon-Teong Teoh  1 , Kim-Ling Chin  2   3 , Nur-Izyan Samsudin  2 , Shih-Keng Loong  2 , Sing-Sin Sam  2 , Kim-Kee Tan  2 , Chee-Sieng Khor  2 , Juraina Abd-Jamil  2 , Nurhafiza Zainal  4 , Annelies Wilder-Smith  5   6 , Keivan Zandi  4   7 , Sazaly AbuBakar  8   9
Affiliations

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

Boon-Teong Teoh et al. BMC Infect Dis. .

Abstract

Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.

Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.

Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001).

Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Keywords: Diagnostics; Infectious disease; Mosquito; RT-LAMP; Vector; Vector-borne; ZIKV.

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Conflict of interest statement

All authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Amplification curves of the ZIKV RT-LAMP assay. A-D, four ZIKV strains; E-M, four DENV, JEV, LGTV, SINV, CHIKV and GETV, respectively; N, negative control; O, y- and x-axis titles
Fig. 2
Fig. 2
Time threshold of positivity for RT-LAMP assays of serially diluted ZIKV RNA. The mean of Tt-values was calculated with available positive results out of four replicates. Error bars indicate the standard deviations of Tt-values from the mean
Fig. 3
Fig. 3
Detection limit of the ZIKV RT-LAMP assay. The probit regression curve was obtained from four replicates of ZIKV RNA in four dilutions (1000, 100, 10, and 1 copy numbers)
Fig. 4
Fig. 4
ZIKV RNA copy numbers of the simulated clinical human saliva, urine and serum specimens that tested positive by qRT-PCR according to the infectious virus titer (n = 10). The dashed line indicates the detection limit of qRT-PCR. The error bars indicate the standard deviation of the viral RNA copy numbers from the mean. Open circle, positive by qRT-PCR only; filled circle, positive by both qRT-PCR and RT-LAMP
See this image and copyright information in PMC

References

    1. Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952;46(5):509–520. doi: 10.1016/0035-9203(52)90042-4. - DOI - PubMed
    1. Fagbami AH. Zika virus infections in Nigeria: virological and seroepidemiological investigations in Oyo state. J Hyg (Lond) 1979;83(2):213–219. doi: 10.1017/S0022172400025997. - DOI - PMC - PubMed
    1. Macnamara FN. Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg. 1954;48(2):139–145. doi: 10.1016/0035-9203(54)90006-1. - DOI - PubMed
    1. Olson JG, Ksiazek TG, Suhandiman, Triwibowo Zika virus, a cause of fever in Central Java, Indonesia. Trans R Soc Trop Med Hyg. 1981;75(3):389–393. doi: 10.1016/0035-9203(81)90100-0. - DOI - PubMed
    1. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, et al. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 2009;360(24):2536–2543. doi: 10.1056/NEJMoa0805715. - DOI - PubMed

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