Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples

• Published in: PloS one Vol. 12; no. 1; p. e0169125
• Main Authors: Sun, Xi-Meng, Ji, Yong-Sheng, Liu, Xian-Yong, Xiang, Mei, He, Guang, Xie, Li, Suo, Jing-Xia, Suo, Xun
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 05.01.2017; Public Library of Science (PLoS)
• Subjects: Animals; Biology and Life Sciences; Blood; Blood tests; Cercopithecus aethiops; Conserved sequence; Cross-reactivity; Deoxyribonucleic acid; Developing countries; Diagnosis; Diagnostic systems; DNA; DNA, Protozoan - analysis; DNA, Protozoan - genetics; Gene amplification; Humans; Laboratories; LDCs; Medicine and Health Sciences; Methods; Nucleic Acid Amplification Techniques - methods; Parasites; Physical Sciences; Polymerase Chain Reaction; Primers; Protozoa; Research and Analysis Methods; Toxoplasma - genetics; Toxoplasma - isolation & purification; Toxoplasma gondii; Toxoplasmosis; Toxoplasmosis, Animal - diagnosis; Toxoplasmosis, Animal - parasitology; Vero Cells; Veterinary colleges; Zoonoses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0169125

Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries.