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Updated: Jan 30, 2026
On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System
Published on: February 23, 2018
[Comparative analysis of LAMP and Real Time PCR methods to detect pathogens of glanders and meliodosis.]
I Yu Shchit1, K B Ignatov2,3, S F Biketov1
1State Scientific Center of Applied Microbiology and Biotechnology, Obolensk, Russia.
Abstract:
Results of detection of Burkholderia mallei and Burkholderia pseudomallei DNA strains by LAMP (Loop-mediated Isothermal Amplification) and Real Time PCR are shown. It has been revealed that, in Real Time PCR, primers steadily detected DNA of those microorganism for the sequences of which they were designed. The above mentioned primers did not detect DNA of heterologous strains. During LAMP method no set of primers showed high analytical sensitivity and specificity. Primers did not detected DNA of all the strains under research to target genes of which they were not intended, but they were capable of directing the synthesis of fragments of genes of heterologous strains. Furthermore, it was difficult to reach the same results during repeated experiments. Failures during LAMP may occur due to existence of GC-reach regions in Burkholderia mallei and Burkholderia pseudomallei genomes and due to emergence of secondary structures in isothermical conditions. It is recommended to use Real Time PCR in order to detect pathogens, in case of such matrixes as Burkholderia mallei and Burkholderia pseudomallei DNAs which are very complicated for LAMP.
Insights
Real Time PCR reliably detects Burkholderia mallei and Burkholderia pseudomallei DNA. Loop-mediated Isothermal Amplification (LAMP) showed poor sensitivity and specificity for these complex bacterial DNA targets.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Accurate detection of Burkholderia mallei and Burkholderia pseudomallei is crucial for diagnostics.
- Loop-mediated Isothermal Amplification (LAMP) and Real Time PCR are common molecular detection methods.
- The complexity of bacterial genomes can impact assay performance.
Purpose of the Study:
- To compare the analytical sensitivity and specificity of LAMP and Real Time PCR for detecting Burkholderia mallei and Burkholderia pseudomallei DNA.
- To evaluate the reliability of these methods when applied to complex DNA matrices.
Main Methods:
- DNA extraction from Burkholderia mallei and Burkholderia pseudomallei strains.
- Design and application of specific primers for LAMP and Real Time PCR assays.
- Performance evaluation based on detection of homologous and heterologous DNA strains.
Main Results:
- Real Time PCR demonstrated high specificity, detecting only target DNA sequences and no heterologous strains.
- LAMP assays exhibited low analytical sensitivity and specificity, with primers amplifying unintended gene fragments from heterologous strains.
- Reproducibility issues were encountered with LAMP, potentially due to GC-rich regions and secondary structures in the target genomes.
Conclusions:
- Real Time PCR is recommended for the reliable detection of Burkholderia mallei and Burkholderia pseudomallei DNA.
- LAMP is not suitable for these complex bacterial DNA targets due to limitations in sensitivity, specificity, and reproducibility.
- Genomic complexity poses challenges for isothermal amplification methods like LAMP.
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