Sensitive and rapid detection of Ortleppascaris sinensis (Nematoda: Ascaridoidea) by loop-mediated isothermal amplification

Abstract

Ortleppascaris sinensis is the dominant nematode species infecting the gastrointestinal tract of the captive Chinese alligator, a critically endangered species. Gastrointestinal nematode infection may cause a loss of appetite, growth, a development disorder, and even mortality in alligators, especially young ones. This research first establishment a loop-mediated isothermal amplification (LAMP) assay in rapidly identifying O. sinensis, upon the basis of the complete internal transcribed spacers (ITS) gene. Eight sets of primers were designed for recognition of the unique conserved ITS gene sequences, and one set was selected to be the most suitable primer for rapid detection. The specific as well as the sensitive features of the most appropriate primer in LAMP reactions for O. sinensis, and feces specimens of Chinese alligators suffering from O. sinensis were determined. Turbidity monitoring and Te Visual Reagent methods were used for determining negative and positive consequences. According to this study, amplification and visualization of the target DNA could be realized through two detection approaches during 50 min at 65 °C isothermal temperature. The sensitivity of LAMP was a detecting limitation of 3.46 pg/µl DNA. No cross-reactions were found between O. sinensis and any other of the nine heterologous nematode parasites, which shows the outstanding specific features of the primers. The LAMP assay could also perform a detection of target DNA of O. sinensis in the feces samples of Chinese alligators. This LAMP assay is useful for directly detecting O. sinensis in the Chinese alligator breeding centers, particularly due to its rapidity, simplicity and low cost.

Rapid and convenient detection of SARS-CoV-2 using a colorimetric triple-target reverse transcription loop-mediated isothermal amplification method

“…To optimize the RT-LAMP reaction, different reaction times (ranging from 30 to 60 min) were executed in the real-time RT-LAMP assays, using 4 copies of SARS-CoV-2 genome RNA as the template, positive signal shown after 30-min reaction time, and NTC shown positively in 45-min reaction time, so 35-min was selected as the optimal reaction time in order to reduce false positives ( Supplemental 2 ) ( Zhao et al, 2019 ).…”

“…Furthermore, the results of the LAMP reaction can be observed by easy-to-see color changes ( Goto et al, 2009 ; Rabe & Cepko, 2020 ; Tanner, Zhang & Evans Jr, 2015 ). Last but not least, LAMP assays have been successfully used for detecting emerging pathogens, such as parasites (Ortleppascaris sinensis) ( Zhao et al, 2019 ); bacteria (TB, and Salmonella) ( WHO, 2016 ; Kim et al, 2022 ); and viruses (HIV, MERS-CoV, and SARS-CoV) ( Kim et al, 2019 ; Lee et al, 2017 ; Li et al, 2019 ). Therefore, RT-LAMP assays are of great value for the screening of SARS-CoV-2 in places such as outpatient clinics and in the field, especially in resource-limited regions.…”

Highly Sensitive Detection of Human Pluripotent Stem Cells by Loop-Mediated Isothermal Amplification

“…With RT-LAMP, we can control positive/negative cutoff copy number of target RNA per test between approximately 10 and 1,000 copies per test by adjusting the reaction buffer (Supplementary Fig. 7 ) and temperature conditions [ 42 ]. This adjustability of the qualitative LAMP cutoff would enable 0% hiPSC contamination to be distinguished as negative and 0.001% contamination as positive based on several marker genes if the RT-LAMP assays were performed with 5 µg total RNA per test (Supplementary Fig.…”

Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2

“…This method is suitable for clinical diagnostics in a resource-poor environment. LAMP (and RT-LAMP) are commonly used in diagnostic microbiological fields to detect pathogens such as viruses (HIV [ 20 ], SARS-CoV-1 [ 21 ] and MERS-CoV [ 22 ]), bacteria (Tuberculosis [ 23 ] and Salmonella [ 24 ]), nosocomial bacteria ( Acinetobacter baumannii ) [ 25 ], fungal pathogens ( Pneumocystis jirovecii ) [ 26 ] and parasites ( Ortleppascaris sinensis [ 27 ] and Phytophthora ramorum [ 28 ]), as well as in the detection of antibiotic-resistant genes (β-lactamases genes [ 29 ]).…”