Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza a H1N1 virus

Manmohan Parida, Jyoti Shukla, Shashi Sharma, Sanna Ranghia Santhosh, Vasanthapuram Ravi, Reeta Mani, Maria Thomas, Shashi Khare, Arvind Rai, Radha Kant Ratho, Sujit Pujhari, Bijayanti Mishra, Putcha Venkata Lakshmana Rao, Rajagopalan Vijayaraghavan

Research output: Contribution to journalArticle

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Abstract

The recent emergence of the swine-origin influenza A H1N1 virus (S-OIV) poses a serious global health threat. Rapid detection and differentiation of S-OIV from seasonal influenza is crucial for patient management and control of the epidemics. A one-step, singletube accelerated and quantitative S-OIV-specific H1 reverse transcription loop-mediated isothermal amplification (RTLAMP) assay for clinical diagnosis of S-OIV by targeting the H1 gene is reported in this article. A comparative evaluation of the H1-specific RTLAMP assay vis-à-vis the World Health Organization-approved real-time polymerase chain reaction (RTPCR), involving 239 acute-phase throat swab samples, demonstrated exceptionally higher sensitivity by picking up all of the 116 H1N1-positive cases and 36 additional positive cases among the negatives that were sequence-confirmed as S-OIV H1N1. None of the real-time RTPCR-positive samples were missed by the RTLAMP system. The comparative analysis revealed that S-OIV RTLAMP was up to tenfold more sensitive than the World Health Organization real-time RTPCR; it had a detection limit of 0.1 tissue culture infectious dosage of 50/ml. One of the most attractive features of this isothermal gene amplification assay is that it seems to have an advantage in monitoring gene amplification by means of SYBR Green I dye-mediated naked-eye visualization within 30 minutes compared to 2 to 3 hours for a real-time reverse transcription polymerase chain reaction. This suggests that the RTLAMP assay is a valuable tool for rapid, real-time detection and quantification of S-OIV in acute-phase throat swab samples without requiring sophisticated equipment.

Original languageEnglish (US)
Pages (from-to)100-107
Number of pages8
JournalJournal of Molecular Diagnostics
Volume13
Issue number1
DOIs
StatePublished - Jan 1 2011

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H1N1 Subtype Influenza A Virus
Human Influenza
Reverse Transcription
Swine
Real-Time Polymerase Chain Reaction
Gene Amplification
Pharynx
Gene Targeting
Limit of Detection
Coloring Agents
Equipment and Supplies
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Molecular Medicine

Cite this

Parida, Manmohan ; Shukla, Jyoti ; Sharma, Shashi ; Santhosh, Sanna Ranghia ; Ravi, Vasanthapuram ; Mani, Reeta ; Thomas, Maria ; Khare, Shashi ; Rai, Arvind ; Ratho, Radha Kant ; Pujhari, Sujit ; Mishra, Bijayanti ; Rao, Putcha Venkata Lakshmana ; Vijayaraghavan, Rajagopalan. / Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza a H1N1 virus. In: Journal of Molecular Diagnostics. 2011 ; Vol. 13, No. 1. pp. 100-107.
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abstract = "The recent emergence of the swine-origin influenza A H1N1 virus (S-OIV) poses a serious global health threat. Rapid detection and differentiation of S-OIV from seasonal influenza is crucial for patient management and control of the epidemics. A one-step, singletube accelerated and quantitative S-OIV-specific H1 reverse transcription loop-mediated isothermal amplification (RTLAMP) assay for clinical diagnosis of S-OIV by targeting the H1 gene is reported in this article. A comparative evaluation of the H1-specific RTLAMP assay vis-{\`a}-vis the World Health Organization-approved real-time polymerase chain reaction (RTPCR), involving 239 acute-phase throat swab samples, demonstrated exceptionally higher sensitivity by picking up all of the 116 H1N1-positive cases and 36 additional positive cases among the negatives that were sequence-confirmed as S-OIV H1N1. None of the real-time RTPCR-positive samples were missed by the RTLAMP system. The comparative analysis revealed that S-OIV RTLAMP was up to tenfold more sensitive than the World Health Organization real-time RTPCR; it had a detection limit of 0.1 tissue culture infectious dosage of 50/ml. One of the most attractive features of this isothermal gene amplification assay is that it seems to have an advantage in monitoring gene amplification by means of SYBR Green I dye-mediated naked-eye visualization within 30 minutes compared to 2 to 3 hours for a real-time reverse transcription polymerase chain reaction. This suggests that the RTLAMP assay is a valuable tool for rapid, real-time detection and quantification of S-OIV in acute-phase throat swab samples without requiring sophisticated equipment.",
author = "Manmohan Parida and Jyoti Shukla and Shashi Sharma and Santhosh, {Sanna Ranghia} and Vasanthapuram Ravi and Reeta Mani and Maria Thomas and Shashi Khare and Arvind Rai and Ratho, {Radha Kant} and Sujit Pujhari and Bijayanti Mishra and Rao, {Putcha Venkata Lakshmana} and Rajagopalan Vijayaraghavan",
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Parida, M, Shukla, J, Sharma, S, Santhosh, SR, Ravi, V, Mani, R, Thomas, M, Khare, S, Rai, A, Ratho, RK, Pujhari, S, Mishra, B, Rao, PVL & Vijayaraghavan, R 2011, 'Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza a H1N1 virus', Journal of Molecular Diagnostics, vol. 13, no. 1, pp. 100-107. https://doi.org/10.1016/j.jmoldx.2010.11.003

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza a H1N1 virus. / Parida, Manmohan; Shukla, Jyoti; Sharma, Shashi; Santhosh, Sanna Ranghia; Ravi, Vasanthapuram; Mani, Reeta; Thomas, Maria; Khare, Shashi; Rai, Arvind; Ratho, Radha Kant; Pujhari, Sujit; Mishra, Bijayanti; Rao, Putcha Venkata Lakshmana; Vijayaraghavan, Rajagopalan.

In: Journal of Molecular Diagnostics, Vol. 13, No. 1, 01.01.2011, p. 100-107.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza a H1N1 virus

AU - Parida, Manmohan

AU - Shukla, Jyoti

AU - Sharma, Shashi

AU - Santhosh, Sanna Ranghia

AU - Ravi, Vasanthapuram

AU - Mani, Reeta

AU - Thomas, Maria

AU - Khare, Shashi

AU - Rai, Arvind

AU - Ratho, Radha Kant

AU - Pujhari, Sujit

AU - Mishra, Bijayanti

AU - Rao, Putcha Venkata Lakshmana

AU - Vijayaraghavan, Rajagopalan

PY - 2011/1/1

Y1 - 2011/1/1

N2 - The recent emergence of the swine-origin influenza A H1N1 virus (S-OIV) poses a serious global health threat. Rapid detection and differentiation of S-OIV from seasonal influenza is crucial for patient management and control of the epidemics. A one-step, singletube accelerated and quantitative S-OIV-specific H1 reverse transcription loop-mediated isothermal amplification (RTLAMP) assay for clinical diagnosis of S-OIV by targeting the H1 gene is reported in this article. A comparative evaluation of the H1-specific RTLAMP assay vis-à-vis the World Health Organization-approved real-time polymerase chain reaction (RTPCR), involving 239 acute-phase throat swab samples, demonstrated exceptionally higher sensitivity by picking up all of the 116 H1N1-positive cases and 36 additional positive cases among the negatives that were sequence-confirmed as S-OIV H1N1. None of the real-time RTPCR-positive samples were missed by the RTLAMP system. The comparative analysis revealed that S-OIV RTLAMP was up to tenfold more sensitive than the World Health Organization real-time RTPCR; it had a detection limit of 0.1 tissue culture infectious dosage of 50/ml. One of the most attractive features of this isothermal gene amplification assay is that it seems to have an advantage in monitoring gene amplification by means of SYBR Green I dye-mediated naked-eye visualization within 30 minutes compared to 2 to 3 hours for a real-time reverse transcription polymerase chain reaction. This suggests that the RTLAMP assay is a valuable tool for rapid, real-time detection and quantification of S-OIV in acute-phase throat swab samples without requiring sophisticated equipment.

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