Multiplex loop‐mediated isothermal amplification assay for the identification of three major whitefly species in the greenhouse

Abstract

A multiplex loop‐mediated isothermal amplification (mLAMP) assay was developed for the identification of three species of whitefly, Trialeurodes vaporariorum, Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1) and Mediterranean (MED), major pests in the greenhouse. Each of the specific LAMP primer sets was designed based on the mitochondrial cytochrome oxidase I (mtCOI) gene sequence. The mLAMP reactions using primer mixtures labelled with fluorescent dye were performed at 63°C for 60 min and centrifuged with polyethyleneimine. Thus, T. vaporariorum, MEAM1 and MED were clearly identified by the colour precipitates under UV light. The mLAMP procedure described in this study is cost‐effective and can be performed in the field not only in the laboratory, because this method is a single analysis and does not need a special gene amplification device.

Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection

“…Sensing and particle surfaces are coated either with streptavidin or with covalently binding, target-specic probes. The insertion of biotin into amplicons by biotin-labelled primers is unproblematic regarding the efficiency of amplication due to Aedes aegypti (Wolbachia infection) 85,86 Ammonia-oxidizing enzyme in environmental bacteria 64 Avian reovirus 70 Bacteriophage MS2 73 Bemisia tabaci 60 BRAF allele (V600E) 83 Brucella 72 Caenorhabditis elegans 68 Chikungunya virus 73 Chlamydia trachomatis 61 Dengue virus 87,112,156 Diarrheal disease 82 Escherichia coli 68 Fomitiporia torreyae 59 Fulviformes umbrinellus 59 Fusarium oxysporum f. sp. lycopersici (point mutations in xylem 3 (SIX3) gene) 66 Genetically modied maize [77][78][79][80][81] Haemophilus ducreyi 101 Haemophilus inuenzae 105 hBRCA1 68 HeLa 68 Hepatitis B and C virus 62,87,107 Herpes simplex virus 1 (HSV1) US4 83 Human immunodeciency virus 87,101 HLA-B*15:02 allele 163 Human papillomavirus 153,171 Human T-lymphotropic virus 1 101 Inuenza virus 63,114 Lambda DNA 68 Leptospira 148 Leishmania 155 Listeria ivanovii 103 Listeria monocytogenes 103 Methicillin-resistant Staphylococcus aureus (MRSA) …”

“…Furthermore, the same method was applied to the identication of three species of whitey, Trialeurodes vaporariorum, Bemisia tabaci Middle East-Asia Minor 1 (MEAM1), and Mediterranean (MED). 60 All three targets were identied with the naked eye in a multiplex assay by the three different colours of the precipitate. 4.2.2.…”

Multiplexing LAMP Assays: A Methodological Review and Diagnostic Application

“…The ability to use different fluorophores has enabled the differentiation of three species of whitefly, Trialeurodes vaporariorum , Bemisia tabaci (MEAM1), and B. tabaci (MED), in a single reaction [ 35 ], and the simultaneous detection of two fungi, Fomitiporia torreyae and Fulviformes umbrinellus [ 36 ]. Despite not inherently facilitating colorimetric result detection, a post-reaction addition of polyethyleneimine (PEI) has been used to precipitate the amplified products after brief centrifugation, resulting in a visually observable pellet under UV light emission [ 35 , 36 ]. To date, this methodology has been employed in various clinical samples (bronchoalveolar lavage fluid, urine, saliva, nasal wash, naso/oropharyngeal swabs) for the detection of varicella-zoster virus [ 34 ].…”

“…The FLOS ( Section 2.1.3 ) [ 35 , 36 ] and assimilation probes ( Section 2.1.5 ) [ 47 ] methodologies have utilized the post-reaction addition of the polymer polyethyleneimine (PEI), which precipitates the amplification products after brief centrifugation, generating a colored pellet visible to the naked eye under UV light. On the other hand, simple methods such as agarose electrophoresis can be used to visualize amplification results obtained with molecular beacon methodology ( Section 2.2.2 ) [ 61 ] or with LAMP products subsequently treated with enzymatic digestion as in the case of methods using endonucleases ( Section 2.3 ) [ 67 , 68 , 69 ].…”

Development of a Rapid, Accurate, and On-Site Detection Protocol for Red Imported Fire Ants, Solenopsis invicta (Hymenoptera: Formicidae)

“…Various nucleic acid amplification methods have been developed to overcome the disadvantages of PCR-based molecular diagnostics. Loop-mediated isothermal amplification (LAMP) is the most widely used method for detecting viruses, microorganisms, nematodes, and insects because it shows outstanding sequence specificity and sensitivity [ 13 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ].…”