A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses.

Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species Human adenovirus B (HAdV-B), Human adenovirus C (HAdV-C) and Human adenovirus E (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90-94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

Comparison of Reverse Transcriptase (RT) Activities of Various M-MuLV RTs for RT-LAMP Assays.

Reverse transcriptases (RTs) are a family of enzymes synthesizing DNA using RNA as a template and serving as indispensable tools in studies related to RNA. M-MuLV RT and its analogs are the most commonly used RTs. RTs are widely applied in various diagnostics methods, including reverse-transcription loop-mediated isothermal amplification (RT-LAMP). However, the performance of different RTs in LAMP remains relatively unknown. Here, we report on the first direct comparison of various M-MuLV RTs in RT-LAMP, including enzymes with a different number of mutations and fusions with Sto7d. Several parameters were assessed, namely: optimal reaction temperature, enzyme concentration, reverse transcription time, a minimal amount of RNA template, and tolerance to inhibitors. Mutations increased the optimal reaction temperature from 55 °C to 60-65 °C. All of the RTs were suitable for RT-LAMP with RNA templates in the range of 101-106 copies per reaction. Highly mutated enzymes were 1.5-3-fold more tolerant to whole blood, blood plasma, and guanidinium, but they were two-fold more sensitive to high concentrations of NaCl. The comparison of different RTs presented here could be helpful for selecting the optimal enzyme when developing novel LAMP-based diagnostic tests.