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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

Specificity of EBOV NP detection by RT-LAMP.(A) Turbidity was monitored and recorded every 6 s by a Loopamp real-time turbidimeter at 650 nm. (B) Visual detection using a calcein fluorescent detection reagent. Lane 1, positive control (artificial EBOV RNA); lane 2, negative control (double-distilled water); lane 3, Sudan EBOV (artificial Sudan EBOV RNA); lane 4, MARV (artificial MARV RNA); lane 5, SARS coronavirus; lane 6, H7N9; lane 7, H1N1; lane 8, H2N3; lanes 9–12, human parainfluenza viruses (PIV) 1, 2, 3, and 4; lanes 13–15, adenoviruses (ADV; serotypes 3, 5, and 55); lanes 16 and 17, respiratory syncytial virus infection, RSVA, RSVB; lane 18, MERS RNA; lane 19, human metapneumovirus, HMPV; lane 20, bocavirus, BoV; lanes 21–24, human coronavirus, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1; lane 25, Legionella pneumophila 9135; lane 26, Mycobacterium tuberculosis 005; and lane 27, Haemophilus influenza ATCC 49247.
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Figure 2: Specificity of EBOV NP detection by RT-LAMP.(A) Turbidity was monitored and recorded every 6 s by a Loopamp real-time turbidimeter at 650 nm. (B) Visual detection using a calcein fluorescent detection reagent. Lane 1, positive control (artificial EBOV RNA); lane 2, negative control (double-distilled water); lane 3, Sudan EBOV (artificial Sudan EBOV RNA); lane 4, MARV (artificial MARV RNA); lane 5, SARS coronavirus; lane 6, H7N9; lane 7, H1N1; lane 8, H2N3; lanes 9–12, human parainfluenza viruses (PIV) 1, 2, 3, and 4; lanes 13–15, adenoviruses (ADV; serotypes 3, 5, and 55); lanes 16 and 17, respiratory syncytial virus infection, RSVA, RSVB; lane 18, MERS RNA; lane 19, human metapneumovirus, HMPV; lane 20, bocavirus, BoV; lanes 21–24, human coronavirus, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1; lane 25, Legionella pneumophila 9135; lane 26, Mycobacterium tuberculosis 005; and lane 27, Haemophilus influenza ATCC 49247.

Mentions: To test the LAMP specificity for NP, we tested 26 non- Zaire EBOV viruses in addition to EBOV itself and in vitro transcribed artificial EBOV RNA as the positive control. Figure 2 shows that EBOV RNA was identified positively by RT-LAMP with the EBL-2 primer set using turbidity monitoring and visual observation. All non- Zaire EBOV strains tested negative, including the blank control, indicating that the RT-LAMP method was specific for EBOV.


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)

Specificity of EBOV NP detection by RT-LAMP.(A) Turbidity was monitored and recorded every 6 s by a Loopamp real-time turbidimeter at 650 nm. (B) Visual detection using a calcein fluorescent detection reagent. Lane 1, positive control (artificial EBOV RNA); lane 2, negative control (double-distilled water); lane 3, Sudan EBOV (artificial Sudan EBOV RNA); lane 4, MARV (artificial MARV RNA); lane 5, SARS coronavirus; lane 6, H7N9; lane 7, H1N1; lane 8, H2N3; lanes 9–12, human parainfluenza viruses (PIV) 1, 2, 3, and 4; lanes 13–15, adenoviruses (ADV; serotypes 3, 5, and 55); lanes 16 and 17, respiratory syncytial virus infection, RSVA, RSVB; lane 18, MERS RNA; lane 19, human metapneumovirus, HMPV; lane 20, bocavirus, BoV; lanes 21–24, human coronavirus, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1; lane 25, Legionella pneumophila 9135; lane 26, Mycobacterium tuberculosis 005; and lane 27, Haemophilus influenza ATCC 49247.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Specificity of EBOV NP detection by RT-LAMP.(A) Turbidity was monitored and recorded every 6 s by a Loopamp real-time turbidimeter at 650 nm. (B) Visual detection using a calcein fluorescent detection reagent. Lane 1, positive control (artificial EBOV RNA); lane 2, negative control (double-distilled water); lane 3, Sudan EBOV (artificial Sudan EBOV RNA); lane 4, MARV (artificial MARV RNA); lane 5, SARS coronavirus; lane 6, H7N9; lane 7, H1N1; lane 8, H2N3; lanes 9–12, human parainfluenza viruses (PIV) 1, 2, 3, and 4; lanes 13–15, adenoviruses (ADV; serotypes 3, 5, and 55); lanes 16 and 17, respiratory syncytial virus infection, RSVA, RSVB; lane 18, MERS RNA; lane 19, human metapneumovirus, HMPV; lane 20, bocavirus, BoV; lanes 21–24, human coronavirus, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1; lane 25, Legionella pneumophila 9135; lane 26, Mycobacterium tuberculosis 005; and lane 27, Haemophilus influenza ATCC 49247.
Mentions: To test the LAMP specificity for NP, we tested 26 non- Zaire EBOV viruses in addition to EBOV itself and in vitro transcribed artificial EBOV RNA as the positive control. Figure 2 shows that EBOV RNA was identified positively by RT-LAMP with the EBL-2 primer set using turbidity monitoring and visual observation. All non- Zaire EBOV strains tested negative, including the blank control, indicating that the RT-LAMP method was specific for EBOV.

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