Future Perspectives on Nucleic Acid Testing
Attila Lorincz in Nucleic Acid Testing for Human Disease, 2016
Another nucleic acid amplification method, loop-mediated isothermal amplification (LAMP), amplifies DNA under isothermal conditions.59 It uses a DNA polymerase and a set of four primers designed to recognize six sequences on the target DNA. The LAMP product has a stem–loop structure and approximately 109 copies can be synthesized in <1 hr. LAMP has been applied to the detection of a range of infectious agents such as human herpes virus 6 (HHV-6) and could detect 25 copies per tube.60 The LAMP procedure produces large amounts of pyrophosphate, and the turbidity produced when pyrophosphate reacts with magnesium ions provides a simple way to monitor the reaction in real time.61
Distribution
Paul Pumpens in Single-Stranded RNA Phages, 2020
Meanwhile, the heterogeneous asymmetric recombinase polymerase amplification (haRPA) was applied for the phage MS2 detection in combination with the stepwise phage concentration from 1250 L drinking water into 1 mL (Elsäßer et al. 2018). Since then, digital PCR (dPCR) has become a promising technology for absolute quantification of nucleic acid without need of calibration curves. Following dPCR, various digital isothermal amplification methods were also developed which only required isothermal incubation. Among them, a loop-mediated isothermal amplification, or LAMP, became the most popular one and was adapted to the rapid enumeration of the phage MS2 as a model (Huang X et al. 2018; Lin et al. 2019).
Clostridium
Dongyou Liu in Handbook of Foodborne Diseases, 2018
Among many rapid protocols for detection of C. botulinum, PCR targeting the BoNT genes is of diagnostic value. Use of multiplex PCR assays enables the simultaneous detection of two or more types of BoNT genes95–97 and discrimination among BoNT serotypes A, B, E, and F, corroborating mouse bioassay results98,99 in the diagnosis of some cases of infant botulism. Fakruddin et al.100 described improvements and alternatives to the PCR such as loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), and rolling circle amplification (RCA).
Loop-mediated isothermal amplification assay as a point-of-care diagnostic tool for Vibrio parahaemolyticus: recent developments and improvements
Published in Expert Review of Molecular Diagnostics, 2019
Karanth Padyana Anupama, Anirban Chakraborty, Iddya Karunasagar, Indrani Karunasagar, Biswajit Maiti
Loop-mediated isothermal amplification (LAMP) is a nucleic acid-based technique that enables DNA amplification under isothermal conditions. It requires a set of four specially designed primers that recognize six distinct regions of the target, and a Bst DNA polymerase with strand displacement activity [27–30]. LAMP assay has the potential to transform the landscape of the existing molecular-based diagnostic techniques. The assay can be done without any sophisticated equipment, without compromising the specificity and sensitivity levels. LAMP is capable of producing a large number of amplicons within an hour [27], and hence, it can be used as the point-of-care diagnostic technique for the identification of foodborne pathogens [28,31–33]. The assay can be carried out directly by eliminating the DNA extraction step [34–36], which has almost no impact as far as the sensitivity of the assay is concerned [27,37]. Unlike PCR assay, the LAMP assays are generally resistant to inhibitors like blood, serum and food ingredients [37–39]. LAMP assays are more sensitive and accurate compared to PCR assays including qPCR [29–32,40,41].
The mycobiota of the human body: a spark can start a prairie fire
Published in Gut Microbes, 2020
Di Zhang, Ying Wang, Sunan Shen, Yayi Hou, Yugen Chen, Tingting Wang
The above methods cannot address the complexity of the fungal community. Therefore, molecular biological methods have entered the stage of fungal detection.185 Polymerase chain reaction (PCR) is a basic technology related to fungal DNA detection. Although traditional PCR technology has advantages of being quick and intuitive, the low DNA content of fungal pathogens has led to the fact that this technique cannot meet the required sensitivity.186 Thus, some improved technologies were born. qPCR allowed us to access real-time detection, enhancing sensitivity and specificity.187 This instant feedback signal was accomplished by fluorescent probes such as Taqman. Isothermal PCR eliminates the thermal cycle step, simplifying the PCR technology greatly. Loop-mediated isothermal amplification has an absolute advantage.188 Use of PCR necessitates attention to some points: DNA extraction should be performed with attention to DNA quality and should avoid contamination;189 in regard to the design of various primers,4 the target is usually directed to the fungal rDNA operon region.190 Researchers have harmonized the biomarkers used to identify fungal species. Based on the considerable sequences and analyses, The taxonomic and phylogenetic classification based on sequence analysis of the ITS genomic region has become a crucial component of fungal ecology and diversity studies.191
Rapid specific detection of oral bacteria using Cas13-based SHERLOCK
Published in Journal of Oral Microbiology, 2023
Jett Liu, Camden Carmichael, Hatice Hasturk, Wenyuan Shi, Batbileg Bor
Limitations of our study included the testing of clinically healthy subjects without comparison to diseased patients. For this study, which was focused on establishing sensitive and specific detection, we reasoned that using healthy subject samples was sufficient. In addition, we observed that not all crRNA-primer sets displayed the same level of fluorescence when in the presence of equal amounts of target gDNA or live bacteria. These data suggest that in the case of gDNA detection, some crRNA-primer sets may display a higher efficiency than others. This could be due to RPA amplification, T7 transcription, crRNA detection, or some combination of the three steps. Further optimization of our crRNA-primer sets may prove fruitful in achieving higher fluorescent signals and faster detection times. Future studies will focus on testing additional crRNA-primer sets for each bacterial target. In addition, alternative isothermal amplification techniques to RPA, such as loop-mediated isothermal amplification [57] may display higher efficiencies and specificity.
Related Knowledge Centers
- DNA
- Polymerase
- Polymerase Chain Reaction
- Pyrophosphate
- Reverse Transcriptase
- Sybr Green I
- Reverse Transcription Loop-Mediated Isothermal Amplification
- Thermal Cycler
- Primer
- Calcein