Challenging TaqMan probe-based real-time PCR and loop-mediated isothermal amplification (LAMP): the two sensitive molecular techniques for the detection of toxoplasmosis, a potentially dangerous opportunistic infection in immunocompromised patients
| Autor: | Ebrahim Mirzajani, Shirzad Fallahi, Nozhat Zebardast, Kourosh Manouchehri Naeini, Bahram Soltani Tehrani, Vajiheh Eskandari, Mansureh Safari Kavishahi, Mohammad Reza Mahmoudi |
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| Rok vydání: | 2020 |
| Předmět: |
Serial dilution
Loop-mediated isothermal amplification Opportunistic Infections Real-Time Polymerase Chain Reaction Sensitivity and Specificity Biochemistry Microbiology Serology Immunocompromised Host 03 medical and health sciences parasitic diseases Genetics TaqMan Animals Humans Medicine Molecular Biology 030304 developmental biology 0303 health sciences biology 030306 microbiology business.industry Toxoplasma gondii General Medicine DNA Protozoan medicine.disease biology.organism_classification DNA extraction Virology Toxoplasmosis Real-time polymerase chain reaction Parasitology business Nucleic Acid Amplification Techniques Toxoplasma |
| Zdroj: | Archives of Microbiology. 202:1881-1888 |
| ISSN: | 1432-072X 0302-8933 |
| DOI: | 10.1007/s00203-020-01903-1 |
| Popis: | Due to defects and drawbacks of most conventional diagnostic methods including serology for the diagnosis of toxoplasmosis as a dangerous opportunistic infection in immunocompromised individuals, the accurate, rapid, and sensitive detection of infection in such patients is essential. In this study, the TaqMan probe-based real-time PCR and, a relatively new nucleic acid amplification method, the loop-mediated isothermal amplification (LAMP) technique was compared based on the repetitive elements (RE) sequence to detect Toxoplasma gondii (T. gondii) DNA in blood samples of immunocompromised individuals. During this study, 119 blood samples from immunocompromised cancer patients with renal failure, undergoing dialysis were studied. After DNA extraction from blood samples using the salt extraction method, the molecular techniques of TaqMan probe-based real-time PCR and LAMP were used to investigate the contamination of the samples with T. gondii, based on the 529 bp (RE) sequence of T. gondii. The analytical sensitivity of LAMP and real-time PCR was evaluated by duplicating the five-step serial dilutions of T. gondii tachyzoites from 0.25 to 5×105 spiked tachyzoites per milliliter of the Toxoplasma seronegative blood sample. The extracted DNA from other parasites and human chromosomal DNA were used to determine the specificity of the molecular methods. The obtained results were analyzed using Kappa statistical test and SPSS22 software. Out of 119 studied samples, 7 (5.8%) and 5 (4.2%) samples were positive for Toxoplasma by TaqMan probe-based real-time PCR and LAMP, respectively. The limits of detection of TaqMan probe-based real-time PCR and RE-LAMP in negative serum samples were one and five tachyzoites (CT 38), respectively. Both real-time PCR and LAMP methods were 100% specific for Toxoplasma detection. Positive results were obtained only with T. gondii DNA, while other DNA samples were negative. The TaqMan probe-based real-time PCR based on the RE sequence showed higher sensitivity to T. gondii DNA detection in blood samples of cancer patients and serial dilutions of parasitic tachyzoites. The results show that TaqMan probe-based real-time PRC is a sensitive and specific method for the detection of toxoplasmosis in immunocompromised individuals, as well as the LAMP assay, which can be used as a suitable alternative diagnostic method for the detection of toxoplasmosis in such patients, without need the for any expensive equipment. |
| Databáze: | OpenAIRE |
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