Abstract
We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 × 106 to 2.2 × 108 IMNV copy numbers μL-1 RNA, and it was similar with the standard RNA extraction (from 1.2 × 106 to 6.3 × 107 IMNV copy numbers μL-1 RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 911-924 |
| Number of pages | 14 |
| Journal | Journal of Fish Diseases |
| Volume | 32 |
| Issue number | 11 |
| DOIs | |
| State | Published - Nov 2009 |
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Keywords
- Infectious myonecrosis virus
- Lateral flow assay
- Litopenaeus vannamei
- Nucleic acid lateral flow method
- Reverse-transcription loop-mediated isothermal amplification
ASJC Scopus subject areas
- Aquatic Science
- veterinary (miscalleneous)
Cite this
Development of a method for the detection of infectious myonecrosis virus by reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow hybrid assay. / Andrade, T. P D; Lightner, Donald V.
In: Journal of Fish Diseases, Vol. 32, No. 11, 11.2009, p. 911-924.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development of a method for the detection of infectious myonecrosis virus by reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow hybrid assay
AU - Andrade, T. P D
AU - Lightner, Donald V
PY - 2009/11
Y1 - 2009/11
N2 - We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 × 106 to 2.2 × 108 IMNV copy numbers μL-1 RNA, and it was similar with the standard RNA extraction (from 1.2 × 106 to 6.3 × 107 IMNV copy numbers μL-1 RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.
AB - We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 × 106 to 2.2 × 108 IMNV copy numbers μL-1 RNA, and it was similar with the standard RNA extraction (from 1.2 × 106 to 6.3 × 107 IMNV copy numbers μL-1 RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.
KW - Infectious myonecrosis virus
KW - Lateral flow assay
KW - Litopenaeus vannamei
KW - Nucleic acid lateral flow method
KW - Reverse-transcription loop-mediated isothermal amplification
UR - http://www.scopus.com/inward/record.url?scp=70350136473&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=70350136473&partnerID=8YFLogxK
U2 - 10.1111/j.1365-2761.2009.01072.x
DO - 10.1111/j.1365-2761.2009.01072.x
M3 - Article
C2 - 19531063
AN - SCOPUS:70350136473
VL - 32
SP - 911
EP - 924
JO - Journal of Fish Diseases
JF - Journal of Fish Diseases
SN - 0140-7775
IS - 11
ER -