Consensus RT-nested PCR detection of yellow head complex genotypes in penaeid shrimp

Area of Science:

• Aquatic animal health
• Molecular diagnostics
• Virology

Background:

• Yellow head virus (YHV) complex causes significant losses in shrimp aquaculture globally.
• Existing diagnostic methods for YHV genotypes may lack specificity and sensitivity.
• Accurate detection of distinct YHV genotypes is crucial for disease management.

Purpose of the Study:

• To develop and validate a consensus RT-nested PCR (RT-nPCR) assay for detecting all six YHV complex genotypes.
• To improve diagnostic specificity and sensitivity for YHV detection in Penaeus monodon.
• To compare the performance of the developed assay with existing OIE-recommended PCR tests.

Main Methods:

• Design of consensus primers targeting conserved ORF1b gene regions of YHV complex genotypes.
• Optimization of RT-nested PCR with short amplicon sizes (359 bp for PCR, 147 bp for nPCR) to mitigate RNA degradation.
• Analytical sensitivity testing using known RNA copy numbers.
• Group-specificity testing using RNA from 17 Penaeus monodon shrimp infected with different YHV genotypes.
• Phylogenetic analysis of nPCR amplicon sequences for genotype assignment.

Main Results:

• The consensus RT-nPCR assay successfully detected all six YHV complex genotypes.
• Analytical sensitivity was approximately 1250 RNA copies for PCR and 1.25 RNA copies for nPCR.
• The consensus RT-nPCR demonstrated superior group-specificity compared to other OIE-recommended PCR tests.
• Phylogenetic analysis using nPCR amplicon sequences provided genotype assignments consistent with longer sequences.

Conclusions:

• The developed consensus RT-nPCR is a highly sensitive and specific tool for detecting all YHV complex genotypes.
• This assay offers improved diagnostic capabilities for YHV in Penaeus monodon.
• The method facilitates accurate identification and differentiation of YHV genotypes, aiding in disease surveillance and control.