Reverse dot blot hybridization assay— An expeditious diagnostic tool for drug resistant TB

Tuberculosis (TB) is one of the leading infectious diseases that is highly transmissible. It is reported to cause approximately 10 million infections and 1.4 million deaths globally in the year of 2019.¹ Prompt detection and treatment of TB are essential to intercept the proliferation of the disease. In the case of drug- resistant TB, failure to detect the resistance of anti TB drugs may give rise to extensively drug- resistant tuberculosis (XDR-TB). Thus, the rapid detection of drug resistance is vital;, however, the current standard drug susceptibility tests (DST) may take up to 12 weeks raising an alarming concern.² A study was done in China by Li Wan et al, on the accuracy of the reverse dot blot hybridization assay (RDBH) for rapidly detecting the resistance of four anti TB drugs (rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB)) in mycobacterium tuberculosis (MTB) isolates.³ Continuous...

Evaluation of accessible regions of Escherichia coli fimH mRNA through computational prediction and experimental investigation

Background and Objectives: This study aimed to investigate the accessible regions of the fimH mRNA using computational prediction and dot-blot hybridization to increase the effectiveness of antisense anti-virulence therapeutics against Uropatho- genic Escherichia coli. Materials and Methods: We predicted the secondary structure of the E. coli fimH mRNA using the Sfold and Mfold Web servers and RNA structure 5.5 program. Considering the predicted secondary structure, accessible regions in mRNA of fimH were determined and oligonucleotides complementary to these regions were synthesized and hybridization activity of those oligonucleotides to the fimH Digoxigenin (DIG) labeled mRNA was assessed with dot-blot hybridization. Results: When searching the fimH gene in the GenBank database, two lengths for this gene was discovered in different strains of E. coli. The difference was related to the nine bases in the first part of the gene utilizing either of two translation initiation sites. Based on the bioinformatics analyses, five regions lacking obvious stable secondary structures were selected in mRNA of fimH. The result of dot-blot hybridization exhibited strongest hybridization signal between the antisense oli- gonucleotide number one and fimH labeled mRNA, whereas hybridization signals were not seen for the negative control. Conclusion: The results obtained here demonstrate that the region contains start codon of fimH mRNA could act as the potential mRNA target site for anti-fimH antisense therapeutics. It is recommended in the future both of utilizing translation initiation sites be targeted with antisense oligomers compounds.

An Integrated Diagnostic Approach for Citrus Exocortis Viroid

BackgroundCitrus exocortis viroid (CEVd) is a circular single-stranded RNA pathogen consists of around 370 nucleotides and leads to a severe disease showing bark scaling symptom on citrus crops, which leads to yield decrease and economic loss. Since the absence of viroid-encoded proteins, methods for CEVd detection mainly counts on bioassays or nucleic acid-base approaches. In order to validate the CEVd disease, here we developed an integrated diagnostic protocol. MethodsCEVd transcripts were inoculated onto two susceptible cultivars of Solanum lycopersicum L., cv. Rutgers and cv. Double-Fortune, seedings. After inoculation, total RNAs of the two tomato cultivars were extracted to detect CEVd infection by dot blot hybridization, one-step reverse transcription PCR (one-step RT-PCR) and real-time reverse transcription PCR (real-time RT-PCR). In addition, the symptom development of both cultivars was recorded weekly. ResultsThe tomato cultivar Rutgers rather than Double-Fortune or others was selected as a suitable CEVd-indicator plant and the bio-index score was established based on epinasty, vein necrosis, leaf size reduction and stunting symptoms. In addition, the isolate of CEVd that collected from citrus field could rapidly and consistently cause the index symptoms on Rutgers. As expected, CEVd could be specifically and sensitively detected in both tomato and citrus plants by dot-blot hybridization and RT-PCR technologies, including one-step RT-PCR and real-time RT-PCR. Furthermore, we found that the levels of CEVd genomic RNA or CEVd derived small RNAs are correlated to symptom severity. ConclusionsIn this study, we developed an integrated detection method for CEVd and revealed potential underlying viroid-host interactions.

Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization

Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples.