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Survey and Visual Detection of Zaire ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra Leone.

Li H, Wang X, Liu W, Wei X, Lin W, Li E, Li P, Dong D, Cui L, Hu X, Li B, Ma Y, Zhao X, Liu C, Yuan J - Front Microbiol (2015)

Bottom Line: Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates.The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR).Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV.

View Article: PubMed Central - PubMed

Affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.

ABSTRACT
Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus using the nucleoprotein gene (NP) as a target sequence. Two different techniques were used, a calcein/Mn(2+) complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.

No MeSH data available.


Related in: MedlinePlus

Comparison of RT-LAMP sensitivities in detecting EBOV NP. Artificial EBOV RNA was serially diluted 10-fold from 4.56 × 104 copies/μL to 4.56 × 10-2 copies/μL. (A) Turbidity was monitored with a Loopamp Realtime Turbidimeter at 650 nm every 6 s. (B) The reaction was detected visually using a calcein fluorescent detection reagent. Artificial EBOV RNA concentrations were: tube 1, 4.56 × 104 copies/μL; tube 2, 4.56 × 103 copies/μL; tube 3, 4.56 × 102 copies/μL; tube 4, 4.56 × 101 copies/μL; tube 5, 4.56 copies/μL; tube 6, 4.56 × 10-1 copies/μL; tube 7, 4.56 × 10-2 copies/μL; tube 8, ddH2O.
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Figure 3: Comparison of RT-LAMP sensitivities in detecting EBOV NP. Artificial EBOV RNA was serially diluted 10-fold from 4.56 × 104 copies/μL to 4.56 × 10-2 copies/μL. (A) Turbidity was monitored with a Loopamp Realtime Turbidimeter at 650 nm every 6 s. (B) The reaction was detected visually using a calcein fluorescent detection reagent. Artificial EBOV RNA concentrations were: tube 1, 4.56 × 104 copies/μL; tube 2, 4.56 × 103 copies/μL; tube 3, 4.56 × 102 copies/μL; tube 4, 4.56 × 101 copies/μL; tube 5, 4.56 copies/μL; tube 6, 4.56 × 10-1 copies/μL; tube 7, 4.56 × 10-2 copies/μL; tube 8, ddH2O.

Mentions: To determine the sensitivity of the RT-LAMP assay for EBOV, a series of dilutions were prepared of artificial EBOV RNA ranging from 4.56 × 104 to 4.56 × 10-2 copies/μL. As shown in Figure 3A, the times of positivity detection ranged from 18 min for 4.56 × 104 copies/μL to 36 min for 4.56 copies/μL of virus RNA by real-time monitoring. Thus, the RT-LAMP detection limit for NP is 4.56 copies/μL of artificial RNA in a 61°C reaction lasting for 60 min. For the visual inspection, all positive reactions changed to green while negative ones remained orange under natural or 365 nm UV light (Figure 3B). These data indicate that the sensitivity of the two detection methods was the same. The detection limit of real-time RT-PCR for NP was 4.56 copies/μL, but this was achieved with a higher Ct value (Ct = 41). Thus, we concluded that the sensitivity of the RT-LAMP assay for EBOV was similar or higher than real-time RT-PCR.


Survey and Visual Detection of Zaire ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra Leone.

Li H, Wang X, Liu W, Wei X, Lin W, Li E, Li P, Dong D, Cui L, Hu X, Li B, Ma Y, Zhao X, Liu C, Yuan J - Front Microbiol (2015)

Comparison of RT-LAMP sensitivities in detecting EBOV NP. Artificial EBOV RNA was serially diluted 10-fold from 4.56 × 104 copies/μL to 4.56 × 10-2 copies/μL. (A) Turbidity was monitored with a Loopamp Realtime Turbidimeter at 650 nm every 6 s. (B) The reaction was detected visually using a calcein fluorescent detection reagent. Artificial EBOV RNA concentrations were: tube 1, 4.56 × 104 copies/μL; tube 2, 4.56 × 103 copies/μL; tube 3, 4.56 × 102 copies/μL; tube 4, 4.56 × 101 copies/μL; tube 5, 4.56 copies/μL; tube 6, 4.56 × 10-1 copies/μL; tube 7, 4.56 × 10-2 copies/μL; tube 8, ddH2O.
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Related In: Results  -  Collection

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Figure 3: Comparison of RT-LAMP sensitivities in detecting EBOV NP. Artificial EBOV RNA was serially diluted 10-fold from 4.56 × 104 copies/μL to 4.56 × 10-2 copies/μL. (A) Turbidity was monitored with a Loopamp Realtime Turbidimeter at 650 nm every 6 s. (B) The reaction was detected visually using a calcein fluorescent detection reagent. Artificial EBOV RNA concentrations were: tube 1, 4.56 × 104 copies/μL; tube 2, 4.56 × 103 copies/μL; tube 3, 4.56 × 102 copies/μL; tube 4, 4.56 × 101 copies/μL; tube 5, 4.56 copies/μL; tube 6, 4.56 × 10-1 copies/μL; tube 7, 4.56 × 10-2 copies/μL; tube 8, ddH2O.
Mentions: To determine the sensitivity of the RT-LAMP assay for EBOV, a series of dilutions were prepared of artificial EBOV RNA ranging from 4.56 × 104 to 4.56 × 10-2 copies/μL. As shown in Figure 3A, the times of positivity detection ranged from 18 min for 4.56 × 104 copies/μL to 36 min for 4.56 copies/μL of virus RNA by real-time monitoring. Thus, the RT-LAMP detection limit for NP is 4.56 copies/μL of artificial RNA in a 61°C reaction lasting for 60 min. For the visual inspection, all positive reactions changed to green while negative ones remained orange under natural or 365 nm UV light (Figure 3B). These data indicate that the sensitivity of the two detection methods was the same. The detection limit of real-time RT-PCR for NP was 4.56 copies/μL, but this was achieved with a higher Ct value (Ct = 41). Thus, we concluded that the sensitivity of the RT-LAMP assay for EBOV was similar or higher than real-time RT-PCR.

Bottom Line: Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates.The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR).Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV.

View Article: PubMed Central - PubMed

Affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.

ABSTRACT
Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus using the nucleoprotein gene (NP) as a target sequence. Two different techniques were used, a calcein/Mn(2+) complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.

No MeSH data available.


Related in: MedlinePlus