Amplificação isotérmica mediada por loop para detecção de patógenos de plantas

Disease control is crucial to minimize potential losses in agriculture and thereby maintain high crop yield. However, for its effectiveness, the pathogen must be detected early and correctly in the production fields. Different methods of diagnosis can be used, from those based on symptoms to molecular tests. Loop-mediated isothermal amplification (LAMP) is a molecular technique that has been widely used in several biological fields, due to the ease with which it can be applied. The reaction can be carried out in a single thermal condition, due to the use of Bst DNA polymerase, isolated from the bacterium Bacillus stearothermophilus, which has high displacement activity. LAMP is a highly exponential amplification method that produces the target DNA in amounts 109 -1010 times between 45 and 60 minutes at 60-65°C. Its advantages are the visualization of results directly with the naked eye and the fact that it does not need sophisticated equipment for its application. In phytopathology, the technique has been gaining prominence in the detection of fungi, viruses, bacteria, nematodes and phytoplasmas, as well as in the monitoring of fungicide-resistant fungi. LAMP can benefit agriculture so that early, accurate and sensitive diagnostics can be carried out in the fields of cultivation and minimize losses caused by diseases. In this review, we present and discuss LAMP tests, developed for plant pathogens detection, which can be useful for researchers who wish to use the technique in their research area

A Comparative Study of LAMP and PCR in Relation to Time and Cost

Background: The Loop-mediated isothermal amplification (LAMP) represents a very sensitive, easy to use, and less time consuming diagnostic method. Aims: The aim was to establish a simple, cost-effective, molecular technique. Materials and methods: An analytical study was conducted using two hundred acute serum samples using two different molecular techniques; qPCR and LAMP to standardize a costeffective and less time-consuming technique. Results: The cost of in-house LAMP reagents was one-ninth of the cost of commercial qPCR. Consume cost was 23 times less than qPCR besides, lab setup cost was 92 times less than qPCR. More importantly, LAMP requires 5-6 times less time duration than qPCR. Conclusion: Due to its simple short-time operation with low cost, it would be a prevalent molecular technique globally, particularly in Bangladesh. J Shaheed Suhrawardy Med Coll 2020; 12(2): 72-75

Photocatalytic dye degradation and photoexcited anti-microbial activities of green zinc oxide nanoparticles synthesized via Sargassum muticum extracts

Drug-resistant superbugs (DRS) were isolated from hospital sewage waste and confirmed by a 16S rDNA molecular technique as B. filamentosus, B. flexus, P. stutzeri, and A. baumannii.

Comparative evaluation of cost effective extraction free molecular technique for detection of SARS-CoV-2 with reference to standard VTM based RT-qPCR method

Background and Objectives: The entire globe is undergoing an unprecedented challenge of COVID-19. Considering the need of rapid and accurate diagnostic tests for SARS-CoV-2, this study was planned to evaluate the cost effective extraction free RT-PCR technique in comparison to the standard VTM based RT-qPCR method. Materials and Methods: Paired swabs from nasopharynx and oropharynx were collected for SARS-CoV-2 testing, from 211 adult patients (≥18 years) in VTM and plain sterile tubes (dry swabs). These samples were processed and RT-qPCR was carried out as per standard protocols. Results: 54.5% of the patients were females and 45.5% were males with sex ratio 1:1.19 (M: F). 38.86% were symptomatic, of which fever (86.59%), cough (79.23%) and breathlessness (46.34%) were the most common symptoms. The positivity by VTM based method and index method was 31.27% and 13.27% respectively. Of the 27 inconclusive results from index method, 37.04% were positive, 48.15% were negative by VTM based method. However, in 40 inconclusive results by VTM based method, 90% were negative and rest remained inconclusive by index method. The sensitivity and specificity of the index method were 39.39% and 85.71% respectively. The overall agreement between VTM based method and index method was 49.59% with estimated Kappa value of 0.19. Conclusion: VTM based method showed higher sensitivity compared to the index method. The higher positivity by VTM based method, suggests that VTM based method could plausibly be a better detection method of SARS-CoV-2. Still, the index method might add value in a resource limited setups for detection of SARS-CoV-2.

Phage display – a tool for detection and prevention against pathogens. A review

In this article are reviewed the promising uses of phage display in the areas such as microbial pathogens detection of and vaccination. Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phages thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but have also the capability to replicate. Recent data acquired from genome sequencing and advances in phage biology research have aided the development of phage-derived bacterial detection and treatment strategies.

Evaluating the impacts of metabarcoding primer selection on DNA characterization of diet in an aerial insectivore, the Purple Martin

DNA metabarcoding is a molecular technique frequently used to characterize diet composition of insectivorous birds. However, results are sensitive to methodological decisions made during sample processing, with primer selection being one of the most critical. The most frequently used DNA metabarcoding primer set for avian insectivores is ZBJ. However, recent studies have found that ZBJ produces significant biases in prey classification that likely influence our understanding of foraging ecology. A new primer set, ANML, has shown promise for characterizing insectivorous bat diets with fewer taxonomic biases than ZBJ, but ANML has not yet been used to study insectivorous birds. Here, we evaluate the ANML primer set for use in metabarcoding of avian insectivore diets through comparison with the more commonly used ZBJ primer set. Fecal samples were collected from both adult and nestling Purple Martins (Progne subis subis) at 2 sites in the USA and 1 site in Canada to maximize variation in diet composition and to determine if primer selection impacts our understanding of diet variation among sites. In total, we detected 71 arthropod prey species, 39 families, and 10 orders. Of these, 40 species were uniquely detected by ANML, whereas only 11 were uniquely detected by ZBJ. We were able to classify 54.8% of exact sequence variants from ANML libraries to species compared to 33.3% from ZBJ libraries. We found that ANML outperformed ZBJ for PCR efficacy, taxonomic coverage, and specificity of classification, but that using both primer sets together produced the most comprehensive characterizations of diet composition. Significant variation in both alpha- and beta-diversity between sites was found using each primer set separately and in combination. To our knowledge, this is the first published metabarcoding study using ANML primers to describe avian diet, and also the first to directly compare results returned by ANML and ZBJ primer sets.

ISOLATION AND SCREENING OF PROTEOLYTIC BACTERIA FROM SIDOARJO SHRIMP PASTE AS PROTEASE SOURCE TO EXTRACT THE COLLAGEN FROM MILKFISH SCALES (CHANOS CHANOS)

This research aimed to isolate protease-producing bacteria from Sidoarjo shrimp paste for extracting collagen from milkfish scales. This study began with isolation, followed by screening and purification of protease-producing bacterial isolates. Further confirmation of the isolates’ proteolytic indices and the crude protease production, the enzymes’ efficacy in extracting collagen from milkfish scales were tested, followed by pathogenicity and identification using 16S rRNA molecular technique. The study has successfully isolated 15 proteolytic bacterial isolates using skimmed milk agar, but only isolates of TR-10, TR-4.1.1, and TR-15.1 exhibited prospective proteolytic activity based on their corresponding proteolytic indices of 2.96 ± 0.06, 3.10 ± 0.10, and 3.71 ± 0.48. Although the proteolytic activity of isolates TR-10 (0.22 ± 0.05 U/mL) and TR-15.1 (1.07 ± 0.14 U/mL) was high in a salt medium using peptone as the nitrogen source, only the former showed satisfactory activity to extract soluble collagen from milkfish scales. Based on the 16SrRNA, the TR-10 isolate was identified as Bacillus megaterium. The non-pathogenicity of the TR-10 bacterium signified its promising role as a protease source for the halal collagen extraction from milkfish scales.

Endodontic Recontamination and Retreatment: The Concise Systematic Review

Introduction: It is necessary to know the nature of the endodontic microbiota within the root canal system of teeth with necrotic pulp tissues. There are several methods of microbial identification, including techniques based on culture or non-predominance of facultative anaerobes and Gram-positive species, especially Enterococcus faecalis. The 16S ribosomal RNA (rRNA) gene sequencing approach has become the reference method. Polymerase chain reaction (PCR) is a molecular technique also used. A condition for successful endodontic retreatment is proper cleaning of the root canals. Objective: Evaluate through a systematic literature review the main contaminations, recontaminations, and endodontic retreatments in root canals. Methods: The present study was followed by a systematic literature review model, according to the PRISMA rules. Clinical studies included case reports, retrospective, prospective and randomized trials. The quality of the studies was based on the GRADE instrument. The risk of bias was analyzed according to the Cochrane instrument. Results: A total of 94 articles were found. A total of 58 articles were evaluated in full, and 34 were included and discussed in this study. The overall assessment did not result in significant risks that could compromise the science of the present study. According to the GRADE classification, the studies were of moderate quality. Conclusion: It was concluded that it is essential to characterize the microbiota of root canals with failed endodontic treatment through 16S ribosomal RNA (GS) gene sequencing and PCR. Furthermore, it can be stated that the root canal instrumentation system with rotating files maintains the quality of root preparation, reducing the operative time and also the risk of a torsional fracture within the root canal.

Loop Mediated Isothermal Amplification as a Rapid Molecular Method for Pseudomonas aeruginosa T3SS ExoY Gene Septicemia Detection in Beta- Lactamase Species Co-Infections

Background: Pseudomonas aeruginosa is among the most important causative agent of infection in chronically ill patients admitted in hospitals globally. Coupled with its, mixed symptomatology, rapid drug resistance tendency and its causation of severe disease, a fast, reliable and affordable diagnostic technique is required to enable healthcare providers expeditiously mitigate its progression and eventual treatment. The Loop-Mediated Isothermal Amplification (LAMP) technique has the potential to serve as a simple, rapid, specific, sensitive and cost-effective point-of-care diagnostic tool. Broad Objective: To investigate Loop Mediated Isothermal Amplification as a molecular technique for microbial diagnostic and prognostic predictor. Study Design: This study was aimed at evaluating LAMP assay against Simple Polymerase chain reaction and Multiplex PCR on the diagnosis of P. aeruginosa in mixed clinical samples. Materials and Methods: This study developed P. aeruginosa Loop Mediated Isothermal Amplification (PaLAMP) assay to target the ExoY gene with appropriate primer testing and validation procedures. Culture of patient bacterial samples was done on MHA and MHB medium, grown overnight in an Incubator and a incubating shaker at 37oc respectively. Housekeeping gene were identified through online bioinformatics and blasted against known sequences. A set of 6 primers, comprising 2 outer primers (F3 and B3), 2 inner primers (FIP and BIP), and 2 loop primers (FLP and BLP), were designed. Microbial DNA extraction was done followed by PCR amplification as a classical identification using LAMP outer primers 9(F3 and B3). LAMP amplicons were detected by real time turbidimetry (LA-500) at 64°C for 40 minutes as well as under UV light with 1.0 μl of 1/10-diluted original SYBR Green I. Results: LAMP validation against traditional PCR shows a high limit of detection at 10-6ng/µl compared to 10-5ng/µl for PCR. The findings are consistent with outcomes for real time turbidimetric outcomes. Real time LAMP turbidimetric results was cross validated by direct observation through SYBR fluorescence under UV light for positive P. aeruginosa detection through positive amplification. Conclusion: Thus far, Loop mediated isothermal amplification show significantly high limit of detection comparable to standard PCR, its use in field based diagnosis offers an opportunity for a cheap, reliable and faster method to determine disease trends and therapy approaches. This method can be applied in primary care to enhance accuracy in diagnosis and thereby prompt initiation of mitigation treatment regimens.