Shchit I Yu, Ignatov K B, Biketov S F
State Scientific Center of Applied Microbiology and Biotechnology, Obolensk, Russia.
The Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russia.
Klin Lab Diagn. 2018;63(6):378-384. doi: 10.18821/0869-2084-2018-63-6-378-384.
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.
展示了通过环介导等温扩增法（LAMP）和实时荧光定量聚合酶链反应（Real Time PCR）检测鼻疽伯克霍尔德菌和类鼻疽伯克霍尔德菌DNA菌株的结果。结果表明，在实时荧光定量聚合酶链反应中，引物能稳定地检测出它们所针对序列的那些微生物的DNA。上述引物未检测到异源菌株的DNA。在LAMP方法中，没有一组引物表现出高分析灵敏度和特异性。引物未检测到所有它们并非针对的研究菌株的靶基因的DNA，但它们能够指导异源菌株基因片段的合成。此外，在重复实验中很难得到相同的结果。LAMP过程中出现失败可能是由于鼻疽伯克霍尔德菌和类鼻疽伯克霍尔德菌基因组中存在富含GC的区域以及在等温条件下出现二级结构。对于像鼻疽伯克霍尔德菌和类鼻疽伯克霍尔德菌DNA这样对LAMP来说非常复杂的基质，建议使用实时荧光定量聚合酶链反应来检测病原体。
