Rapid Detection of Zika Virus in Urine Samples and Infected Mosquitos by Reverse Transcription-Loop-Mediated Isothermal Amplification

Abstract

Infection with Zika virus (ZIKV) is of growing concern since infection is associated with the development of congenital neurological disease. Quantitative reverse transcription PCR (qRT-PCR) has been the standard for ZIKV detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper testing. Studies have suggested that ZIKV detection in urine is more sensitive and has a longer window of detection compared to serum and saliva. The objective of this study was to develop a urine diagnostic test that could be completed in under 30 minutes. Urine samples spiked with ZIKV or dengue virus were tested using RT-LAMP as well as by conventional quantitative qRT-PCR. These techniques were then validated using crude lysates made from ZIKV infected mosquitoes in addition to urine and serum samples from ZIKV infected patients. RT-LAMP specifically detected ZIKV in urine and serum for ZIKV infected patients and crude mosquito lysates. This test was performed in under 30 minutes and did not require RNA extraction from urine nor mosquitos. This approach could be used for monitoring of exposed individuals, especially pregnant women, couples wanting to conceive, or individuals with suspicious symptoms as well as surveillance of mosquito populations.

Figure 1

RT-LAMP detection of ZIKV. (A) ZIKA RT-LAMP amplification of ZIKV PCR Standard (ZIKV; Robert Koch Institute) but not no template control (NTC; negative control) as visualized by addition of SYBR Green I (SYBR) by eye (upper panel), green fluorescence (middle panel), or gel electrophoresis (bottom panel). Lane M: Low DNA Mass Marker (ThermoFisher Scientific). (B) All primers (All) are required for effective LAMP reaction. Reactions without FIP and BIP (-FIP/BIP) or FL and BL (-FL/BL) resulted in a negative RT-LAMP reaction.

Figure 2

ZIKV RT-LAMP sensitivity for ZIKV. Sensitivity assessment of ZIKV RT-LAMP using serial dilutions of ZIKV PCR Standard (Robert Koch Institute) from 4 × 10⁵ genome copies/reaction to 1 copies/reaction as visualized by addition of SYBR Green I by eye (upper panel), green fluorescence (middle panel), or gel electrophoresis (bottom panel). Lane M: Low DNA Mass Marker (ThermoFisher Scientific); NTC: No template control (negative control). Relative density (RD) of the entire bands for a column relative to the DNA Mass Marker are indicated below the corresponding lane. Lanes that did not have any detectable bands over background are reported as not detectable (ND).

Figure 3

ZIKV RT-LAMP specificity for ZIKV. Specificity assessment of ZIKV RT-LAMP in ZIKV or DENV infected whole cell lysates (WCL) or cell culture supernatants (SN) as visualized by the addition of SYBR Green I by eye (upper panel), green fluorescence (middle panel), or gel electrophoresis (bottom panel). Lane M: Low DNA Mass Marker (ThermoFisher Scientific); NTC: No template control (negative control); + : ZIKV PCR Standard (Robert Koch Institute; positive control).

Figure 4

ZIKV and 18S rRNA RT-LAMP in simulated urine samples. Urine samples were spiked with either different strains of ZIKV or DENV and subjected to a ZIKV (A) or 18S rRNA (B) specific RT-LAMP reaction then visualized by addition of SYBR Green I by eye (upper panel), green fluorescence (middle panel), or gel electrophoresis (bottom panel). Lane M: Low DNA Mass Marker (ThermoFisher Scientific); NTC: No template control (negative control). Relative density (RD) of the entire bands for a column relative to the DNA Mass Marker are indicated below the corresponding lane. Lanes that did not have any detectable bands over background are reported as not detectable (ND).

Figure 5

ZIKV and Actin detection in mosquitos. (A) qRT-PCR of ZIKV RNA normalized to total RNA for mock (n = 5) or ZIKV (n = 5) infected mosquitos. (B,C) Single mosquitos were infected with either mock, ZIKV, or DENV and subjected to a ZIKV (B) or Aedes aegypti Actin (C) specific RT-LAMP reaction then visualized by addition of SYBR Green I by eye (upper panel), green fluorescence (middle panel), or gel electrophoresis (bottom panel). Lane M: Low DNA Mass Marker (ThermoFisher Scientific); NTC: No template control (negative control). Relative density (RD) of the entire bands for a column relative to the DNA Mass Marker are indicated below the corresponding lane. Lanes that did not have any detectable bands over background are reported as not detectable (ND). (D) Detection of ZIKV or ribosomal s17 by qRT-PCR. Data shown as mean ± standard deviation.

Figure 6

Examination of ZIKV and 18 s rRNA in human clinical samples. (A) ZIKV positive patient samples subjected to a ZIKV specific RT-LAMP reaction without (A) or with (B) RNA isolation then visualized by addition of SYBR Green I by eye (upper panel), green fluorescence (middle panel), or gel electrophoresis (bottom panel). Lane NTC: No template control (negative control); Lane ZIKV: ZIKV PCR Standard (ZIKV; Robert Koch Institute). Relative density (RD) of the entire bands for a column relative to the DNA Mass Marker are indicated below the corresponding lane. Lanes that did not have any detectable bands over background are reported as not detectable (ND). (C) Detection of ZIKV or ribosomal 18 S rRNA by qRT-PCR. Data shown as mean ± standard deviation. (D) ZIKV specific RT-LAMP in asymptomatic control patients.