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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

The most appropriate primers and reaction temperatures for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Turbidity was monitored and recorded every 6 s for five sets of primers used to amplify the target gene with a Loopamp real-time turbidimeter at 650 nm. (A) A total of five sets of primers including EBL-1, EBL-2, EBL-7, EBL-11, and EBL-16 were designed to detect artificial EBOV RNA. (B) Reaction temperatures ranged from 53 to 67°C with 2°C intervals.
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Figure 1: The most appropriate primers and reaction temperatures for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Turbidity was monitored and recorded every 6 s for five sets of primers used to amplify the target gene with a Loopamp real-time turbidimeter at 650 nm. (A) A total of five sets of primers including EBL-1, EBL-2, EBL-7, EBL-11, and EBL-16 were designed to detect artificial EBOV RNA. (B) Reaction temperatures ranged from 53 to 67°C with 2°C intervals.

Mentions: A total of five sets of primers were initially designed to detect artificial EBOV RNA using the Real-time Turbidimeter. As shown in Figure 1A, the EBL-2 primer set amplified NP in the shortest time (∼10 min), so this was chosen as the optimal primer set for RT-LAMP detection of EBOV (Table 1).


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)

The most appropriate primers and reaction temperatures for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Turbidity was monitored and recorded every 6 s for five sets of primers used to amplify the target gene with a Loopamp real-time turbidimeter at 650 nm. (A) A total of five sets of primers including EBL-1, EBL-2, EBL-7, EBL-11, and EBL-16 were designed to detect artificial EBOV RNA. (B) Reaction temperatures ranged from 53 to 67°C with 2°C intervals.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664619&req=5

Figure 1: The most appropriate primers and reaction temperatures for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Turbidity was monitored and recorded every 6 s for five sets of primers used to amplify the target gene with a Loopamp real-time turbidimeter at 650 nm. (A) A total of five sets of primers including EBL-1, EBL-2, EBL-7, EBL-11, and EBL-16 were designed to detect artificial EBOV RNA. (B) Reaction temperatures ranged from 53 to 67°C with 2°C intervals.
Mentions: A total of five sets of primers were initially designed to detect artificial EBOV RNA using the Real-time Turbidimeter. As shown in Figure 1A, the EBL-2 primer set amplified NP in the shortest time (∼10 min), so this was chosen as the optimal primer set for RT-LAMP detection of EBOV (Table 1).

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