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Updated: Jun 2, 2026
Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV
Published on: November 10, 2023
Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians,
G Ravikumar1, S Raje Urs, N B Vijaya Prakash
1Seri-biotech Research Laboratory, Central Silk Board, Carmelaram Post, Kodathi, Bangalore, India. ravikumarpillai@gmail.com
Abstract:
We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794bp DNA fragment from Nosema bombycis, 471bp fragment from B. mori NPV (BmNPV) and 391bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers.
Insights
A new PCR assay detects three major silkworm pathogens: microsporidians, nucleopolyhedrovirus (NPV), and densovirus (DNV). This method offers early, sensitive detection for disease-free silkworm production.
Area of Science:
- * Molecular Biology
- * Entomology
- * Animal Pathology
Background:
- * Silkworm (Bombyx mori) aquaculture is threatened by several major pathogens.
- * Accurate and early detection of these pathogens is crucial for disease management and prevention.
- * Conventional diagnostic methods can be time-consuming and may lack sensitivity.
Purpose of the Study:
- * To develop a novel, rapid, and sensitive PCR-based assay for detecting three key silkworm pathogens.
- * To enable simultaneous detection of microsporidians, nucleopolyhedrovirus (NPV), and densovirus (DNV).
- * To provide a valuable tool for early disease diagnosis in silkworms.
Main Methods:
- * Development of a multiplex PCR assay utilizing three specific primer pairs.
- * Primers designed from conserved regions of microsporidian 16S rRNA, NPV polyhedrin gene, and B. mori densovirus (BmDNV) sequences.
- * Optimization of PCR conditions for specific and sensitive amplification of target pathogen DNA.
Main Results:
- * The multiplex PCR assay successfully generated distinct DNA fragments specific to each pathogen: 794bp for Nosema bombycis, 471bp for B. mori NPV (BmNPV), and 391bp for BmDNV.
- * The assay demonstrated high specificity and sensitivity for detecting pathogenic DNA.
- * The method was validated on other silkworm species, Antheraea mylitta and Samia cynthia ricini, detecting similar pathogens.
Conclusions:
- * The developed PCR assay is a valuable and sensitive tool for the early detection of major silkworm pathogens.
- * This method complements traditional diagnostic techniques, improving disease surveillance.
- * It supports the production and supply of disease-free silkworms, benefiting farmers and the industry.
