BACKGROUND

PCR technique in Vietnam is being applied in many diagnostic laboratories. However, output quality varies between laboratories mainly due to lack of an automatic option, especially in the nucleic acid extraction step. Hence, PCR work results depend not only on the quality of diagnostic kits but also on the skills of technical staffs. Nucleic acid extraction automation is the key for a more robust PCR work, minimizing human errors and enhance PCR result reliability.

AIMS

Production and evaluation of the kit using magnetic silica coated nano-iron beads to extract the nucleic acid from different samples for detecting pathogens using PCR and real-time PCR techniques.

MATERIALS – METHODS

In this study, we designed and manufactured various components for extraction kit, named NKDNARNAprep MAGBEAD, implementing the KingFisher automatic nucleic acid extraction open system (KingFisher, KingFisher ML, KingFisher Duo, KingFisher Flex). These components include: magnetic silica coated nano iron beads, extraction buffers and binding buffers, washing buffers and elution buffers. We also optimized different extraction protocols for both DNA and RNA from different sample types, such as serum, plasma, autopsy, shrimp post, shrimp tissue, clam tissue, etc. We validated the quality of these components by direct comparison on the same samples with other commercially available extraction kit such as BOOM (for DNA extraction), Trizol-LS (for RNA extraction), alkaline heat-shock extraction (for DNA extraction from crustacean / seafood). Additionally, we also compared DNA / RNA extraction from human pathogens (HBV, HCV and MTB) using automatic extraction versus MagnaPure (Roche).

RESULTS - DISCUSSIONS

The results suggest that our automatic extraction kit (namely NKDNARNAprep-MAGBEAD): (1) give the same quality / quantity of extracted nucleic acids as MagnaPure (Roche) for DNA/RNA extraction from human pathogens (HBV, HCV, MTB); (2) can be used in the same DNA/RNA extraction five-step protocol for a maxium duration of 45 minutes; (3) is at least 10-times more sensitive than traditional in-house extraction methods; (4) is simplified into two different protocols for fluid and tissue samples; (5) has a 6-month shelf-life at room temperature.

CONCLUSION

The newly designed and manufactured automatic DNA/RNA extraction kit powered by KingFisher open extraction system is the key for diagnostic quality that can be robustly applied by clinical diagnostic laboratories to fully utilize PCR techniques without having to implement more expensive closed PCR system. Therefore, a solution for current technical errors in diagnosis is guaranteed