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Crop Research

In silico approaches for consensus primer design and validation towards development of rapid diagnostics against the Candidatus Liberibacter species


DOI: 10.31830/2454-1761.2025.CR-1024    | Article Id: CR-1024 | Page : 214-220
Citation :- In silico approaches for consensus primer design and validation towards development of rapid diagnostics against the Candidatus Liberibacter species. Crop Res. 60: 214-220
BUTWALL S AND DAS A das.arunava2022@gmail.com
Address : Faculty of Life Science, Mandsaur University, Mandsaur-458001, Madhya Pradesh, India
Submitted Date : 13-05-2025
Accepted Date : 7-06-2025
Online Published:

Abstract

Citrus greening or Huanglongbing (HLB) is a rapidly spreading disease caused by Candidatus Liberibacter species, which inhabit the phloem of citrus plants. It is primarily transmitted by psyllid insects. Managing HLB is challenging due to the bacterium's phloem-restricted nature, making diagnosis and treatment with antimicrobials difficult. Here in the present study, the bacterial consensus sequences were used for the primer design with In silico approaches and validated by PCR. The study was carried out in the Malwa region of Madhya Pradesh, India, which is known for citrus cultivations and remains affected by the HLB disease. Phylogenetic analysis of 100 Candidatus Liberibacter 16S rRNA sequences from NCBI was conducted using MEGA 6, with Malwa-specific refinement via MAFFT alignment. Eight previously deposited sequences formed the basis for the study. A consensus sequence was derived and used to design region-specific primers with GeneFisher. PCR validation was carried out with standard thermal cycling and gel electrophoresis. Phylogenetic analysis of 100 Candidatus Liberibacter 16S rRNA sequences from NCBI was conducted using Phylogenetic analysis showed high genetic variation among Liberibacter strains, supporting the need for region-specific detection. Eight sequences from the Malwa region were aligned using BioEdit, confirming conserved primer sites. Consensus primers were designed using GeneFisher and optimized for 16S rRNA targets. PCR validation identified 22 positives out of 100 samples, with primer set 2 detecting all target species effectively. The study concludes that primer set 2 enables rapid and accurate detection of HLB-causing bacteria in the Malwa region.

Keywords

Candidatus Liberibacter species consensus sequence. huanglongbing (HLB) primer design primer validation

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