Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate

Prasannakumar, M. K.; Parivallal, P. Buela; Manjunatha, C.; Mahesh, H. B.; Pramesh, D.; Narayan, Karthik S.; Gopal, Venkatesh Babu; Kumari, Priyanka; Puneeth, M. E.; Rangaswamy, K. T.

doi:10.1007/s13313-020-00720-w

Cited by 11 publications

(6 citation statements)

References 30 publications

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“…The LAMP assay is a better alternative to the conventional PCR method owing to it being more reliable, cost-effective, highly sensitive, specific, and rapid than PCR [20]. It was used in this study to attempt early detection of BDB in bananas [21,22]. It is primarily the diagnosis and mitigation of crop damage that would benefit from early detection.…”

Section: Discussionmentioning

confidence: 99%

Development of a Highly Sensitive Loop-Mediated Isothermal Amplification Incorporated with Flocculation of Carbon Particles for Rapid On-Site Diagnosis of Blood Disease Bacterium Banana

et al. 2022

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Bananas are one of the most crucial fruit crops worldwide and significantly contribute to food security in developing countries. However, blood disease of bananas caused by Ralstonia syzygii subspecies celebensensis has become a threat to banana production. Rapid and accurate diagnosis of BDB for on-site detection is pivotal at an early stage for an effective disease control strategy. This study developed LAMP with specific primers targeting BDB, followed by a flocculation assay for visualising positive amplification in the LAMP assay. The assay was sensitive to picogram amounts of gDNA (0.5 pg). LAMP assay on BDB gDNA showed flocculation, but negative results on Fusarium oxysporus cubense and Ralstonia solanacaerum confirming the specificity of the assays. Field testing conducted at MARDI headquarters and Taman Pertanian Universiti discovered that the LAMP-flocculation assays were successful in detecting BDB on symptomatic samples as well as on samples from a healthy plot with no symptom observed at the sampling stage, revealing that this assay can detect BDB at an early infection stage. The validation results showed that the LAMP-flocculation assay was comparable with the PCR technique. This newly developed technique is highly specific and sensitive for the early detection of BDB for the adoption of precautionary control measures.

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Section: Discussionmentioning

confidence: 99%

Development of a Highly Sensitive Loop-Mediated Isothermal Amplification Incorporated with Flocculation of Carbon Particles for Rapid On-Site Diagnosis of Blood Disease Bacterium Banana

et al. 2022

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“…The LAMP assay 13 possesses characteristics such as the non-requirement for a thermocycler, rapid amplification, and high specificity and sensitivity well suited for facilitating point-of-care field applications 19 . Therefore, in the present study, we investigated the applicability of isothermal amplification methods (LAMP and HDA) for sensitive and specific diagnosis of M. oryzae and S. oryzae, two predominant seed-borne pathogens of rice.…”

Section: Discussionmentioning

confidence: 99%

“…The LAMP assay has been widely used to detect viruses 17 , bacteria 18 , 19 , and fungal pathogens 20 due to its high sensitivity and specificity compared to other conventional PCR methods 19 . The HDA assay involves in vivo DNA replication using helicase enzyme 14 , 16 , 21 , 22 and does not require initial heat denaturation and subsequent thermocycling steps as required by PCR.…”

Section: Introductionmentioning

confidence: 99%

LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed

Prasannakumar

Parivallal

Pramesh

et al. 2021

Sci Rep

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Rice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.

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“…However, PCR based diagnostic technique demands a costly thermal cycler, and it takes three hours or more for the complete identification of species ( Manjunatha et al, 2018 , Prasannakumar et al, 2021 , Naganur et al, 2019 ). PCR also requires quality DNA for amplification ( Prasannakumar et al, 2020 , Sunani et al, 2019 ) and compounds like polysaccharides, ethanol, phenol, a few proteins, and proteinases ( Rossen et al, 1992 , Rådström et al, 2004 ), act as PCR inhibitors in inhibiting PCR reactions. Hence, attempts were made to develop an alternative, rapid, sensitive, specific colorimetric molecular detection assay for the diagnosis of cassava mealybug.…”

Section: Introductionmentioning

confidence: 99%

Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero

Shivakumar,

Nanjundaiah,

Thiruvengadam

et al. 2024

Saudi Journal of Biological Sciences

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Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate

Cited by 11 publications

References 30 publications

Development of a Highly Sensitive Loop-Mediated Isothermal Amplification Incorporated with Flocculation of Carbon Particles for Rapid On-Site Diagnosis of Blood Disease Bacterium Banana

Development of a Highly Sensitive Loop-Mediated Isothermal Amplification Incorporated with Flocculation of Carbon Particles for Rapid On-Site Diagnosis of Blood Disease Bacterium Banana

LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed

Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero

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