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Corona virus disease 2019 (COVID-19) which initially started as a cluster of pneumonia cases in the Wuhan city of China has now become a full-blown pandemic. Timely diagnosis of COVID-19 is the key in containing the pandemic and breaking the chain of transmission. In low- and middle-income countries availability of testing kits has become the major bottleneck in testing. Novel methods like pooling of samples are the need of the hour. We undertook this study to evaluate a novel protocol of pooling of RNA samples/elutes in performance of PCR for SARS CoV-2 virus. Extracted RNA samples were randomly placed in pools of 8 on a 96 well plate. Both individual RNA (ID) and pooled RNA RT-qPCR for the screening E gene were done in the same plate and the positivity for the E gene was seen. The present study demonstrated that pool testing with RNA samples can easily detect even up to a single positive sample with Ct value as high as 38. The present study also showed that the results of pool testing is not affected by number of positive samples in a pool. Pooling of RNA samples can reduce the time and expense, and can help expand diagnostic capabilities, especially during constrained supply of reagents and PCR kits for the diagnosis of SARS-CoV-2 infection.

Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time quantitative polymerase chain reaction (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probes for the cytochrome c oxidase (COI) gene for the identification of eight ecologically and economically important freshwater fish species: lake whitefish (Coregonus clupeaformis), yellow perch (Perca flavescens), rainbow smelt (Osmerus mordax), brook trout (Salvelinus fontinalis), smallmouth bass (Micropterus dolomieu), round whitefish (Prosopium cylindraceum), spottail shiner (Notropis hudsonius) and deepwater sculpin (Myoxocephalus thompsonii). In order to identify novel primer-probe sets with maximum species-specificity, two separate primer-probe design criteria were employed. Highest ranked primer-probe sets from both methods were assayed to identify sequences that demonstrated highest specificity. Specificity was determined using control species from same genus and non-target species from different genus. Selected primer-probe sets were optimized for annealing temperature and primer-probe concentrations to identify minimum reagent parameters. The selected primer-probe sets were highly sensitive, with DNA concentrations as low as 1 ng adequate for positive species identification. A decoder algorithm was developed based on the cumulative qPCR results that allowed for full automation of species identification. Blinded experiments revealed that the combination of the species-specific primer/probes sets with the automated species decoder resulted in target species identification with 100% accuracy. We also conducted a cost/time comparison analysis between the qPCR assays established in this study with other species identification methods. The qPCR technique was the most cost-effective and least time consuming method of species identification. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification, and the methodology established in this study can be utilized for various other species identification initiatives.

Although DNA extraction is a crucial step before polymerase chain reaction (PCR), attempts to perform PCR without DNA isolation have been ongoing since the 1990s. While partial success has been achieved with direct conventional PCR, the DNA isolation step has remained indispensable for real-time PCR. Here, we developed a method that does not require complete DNA isolation by lysing EDTA-treated whole blood samples through the application of osmotic pressure and heat, followed by centrifugation to get a clear lysate. Blood samples were mixed with distilled water, incubated at 95 °C for 20 min before centrifugation at 14,000 rpm for 5 min. The resulting lysates were used as templates for real-time PCR. Real-time PCRs were performed under identical conditions with lysates and DNA samples using 9 different primer sets. Target genes were successfully amplified using 1:10 and 1:5 diluted blood lysates both at 60 °C and 61 °C. The PCR efficiency for ACTB and PIK3CA differed by 20% and 14%, respectively, between the DNA samples and blood lysates. We successfully amplified all selected genes using “GG-RT PCR”, greater temperature, greater speed method, without the need for additional enzymes or buffers. GG-RT PCR presents a cost effective option for applications such as SNP analysis and deletion detection if appropriate primer designs are made.

The polymerase chain reaction (PCR), is a widely used, sensitive and reliable method for detecting pathogens. However, technical limitations may restrict its use outside sophisticated laboratories, e.g. for detecting pathogens at the site of a disease outbreak. In this study, real-time PCR reagents specific to four bacteria ( Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and to the Influenza A virus were dried using a vacuum oven drying method. The performance of the dried reagents stored at different temperatures, was monitored using both a standard-size and a portable real-time PCR instrument. The vacuum oven dried real-time PCR reagents were stable and retained the sensitivity for at least 14 months when stored in a refrigerator (+ 4 °C). When stored at room temperature, DNA assays remained stable for at least 10 weeks and Influenza A RNA assay for 3 weeks. These results demonstrate the feasibility of vacuum oven dried real-time PCR reagents and a portable thermocycler for the rapid and reliable detection of pathogens. The drying protocol presented here is cost-effective and easy to use, and could be applied to real-time PCR methods specific to other pathogens as well. In addition, this in-house drying protocol reduces reliance on commercial PCR tests during a time of shortage, such as that experienced during the Corovirus disease (COVID-19) crisis.

The Xpert MTB/RIF test enables rapid detection of tuberculosis (TB) and rifampicin resistance. The World Health Organization recommends Xpert for initial diagnosis in individuals suspected of having multidrug-resistant TB (MDR-TB) or HIV-associated TB, and many countries are moving quickly toward adopting Xpert. As roll-out proceeds, it is essential to understand the potential health impact and cost-effectiveness of diagnostic strategies based on Xpert. We evaluated potential health and economic consequences of implementing Xpert in five southern African countries--Botswana, Lesotho, Namibia, South Africa, and Swaziland--where drug resistance and TB-HIV coinfection are prevalent. Using a calibrated, dynamic mathematical model, we compared the status quo diagnostic algorithm, emphasizing sputum smear, against an algorithm incorporating Xpert for initial diagnosis. Results were projected over 10- and 20-y time periods starting from 2012. Compared to status quo, implementation of Xpert would avert 132,000 (95% CI: 55,000-284,000) TB cases and 182,000 (97,000-302,000) TB deaths in southern Africa over the 10 y following introduction, and would reduce prevalence by 28% (14%-40%) by 2022, with more modest reductions in incidence. Health system costs are projected to increase substantially with Xpert, by US$460 million (294-699 million) over 10 y. Antiretroviral therapy for HIV represents a substantial fraction of these additional costs, because of improved survival in TB/HIV-infected populations through better TB case-finding and treatment. Costs for treating MDR-TB are also expected to rise significantly with Xpert scale-up. Relative to status quo, Xpert has an estimated cost-effectiveness of US$959 (633-1,485) per disability-adjusted life-year averted over 10 y. Across countries, cost-effectiveness ratios ranged from US$792 (482-1,785) in Swaziland to US$1,257 (767-2,276) in Botswana. Assessing outcomes over a 10-y period focuses on the near-term consequences of Xpert adoption, but the cost-effectiveness results are conservative, with cost-effectiveness ratios assessed over a 20-y time horizon approximately 20% lower than the 10-y values. Introduction of Xpert could substantially change TB morbidity and mortality through improved case-finding and treatment, with more limited impact on long-term transmission dynamics. Despite extant uncertainty about TB natural history and intervention impact in southern Africa, adoption of Xpert evidently offers reasonable value for its cost, based on conventional benchmarks for cost-effectiveness. However, the additional financial burden would be substantial, including significant increases in costs for treating HIV and MDR-TB. Given the fundamental influence of HIV on TB dynamics and intervention costs, care should be taken when interpreting the results of this analysis outside of settings with high HIV prevalence.

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 10 7 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.

Background Severe acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD). Methods Nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples. Results Custom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A (H1N1)pdm09 and 94.66 – 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more. Conclusion The custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.

Avian infectious bronchitis virus (IBV) is a highly transmissible virus that causes respiratory, reproductive, and renal syndromes, resulting in significant economic losses for the global poultry industry. This study aimed to evaluate the application of high-resolution melting analysis (HRM) as a novel, efficient, and cost-effective tool for the rapid diagnosis and genotyping of Egyptian IBV strains. The investigation focused on the N gene and the 3’ untranslated region (3’ UTR). HRM curves were generated from 435 bp PCR products obtained from 22 IBV-positive avian samples. The purified PCR products, which amplified the 3’ UTR, were sequenced and subsequently compared with analogous S1 gene sequences to differentiate between vaccine strains and field strains, specifically the Egyptian strain 4/91 (GI-13), variant II (GI-23), and the classic strain (GI-1). The high-resolution melting curve analysis (HRM) successfully distinguished vaccine reference strains (Ma5, IB-Primer, 4/91, and Variant II), while the IBV field strains were categorized into three distinct pattern clusters based on the similarity of their melting curves, which were confirmed by S1 gene sequences. Both strains 4/91 and variant II exhibited a deletion of about 15 nucleotides at the terminus of the N-gene compared to classic strains. Closed-tube PCR combined with HRM-curve analysis provides a fast and reliable technique for genotyping IBV strains, including IBV vaccine strains and IBV field strains.

Oral fluid sampling and testing offers a convenient, unobtrusive mechanism for evaluating the health status of swine, especially grower and finisher swine. This assessment evaluates the potential testing of oral fluid samples with real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) to detect African swine fever, classical swine fever, or foot-and-mouth disease for surveillance during a disease outbreak and early detection in a disease-free setting. We used a series of logical arguments, informed assumptions, and a range of parameter values from literature and industry practices to examine the cost and value of information provided by oral fluid sampling and rRT-PCR testing for the swine foreign animal disease surveillance objectives outlined above. Based on the evaluation, oral fluid testing demonstrated value for both settings evaluated. The greatest value was in an outbreak scenario, where using oral fluids would minimize disruption of animal and farm activities, reduce sample sizes by 23%-40%, and decrease resource requirements relative to current individual animal sampling plans. For an early detection system, sampling every 3 days met the designed prevalence detection threshold with 0.95 probability, but was quite costly. Implementation of oral fluid testing for African swine fever, classical swine fever, or foot-and-mouth disease surveillance is not yet possible due to several limitations and information gaps. The gaps include validation of PCR diagnostic protocols and kits for African swine fever, classical swine fever, or foot-and-mouth disease on swine oral fluid samples; minimal information on test performance in a field setting; detection windows with low virulence strains of some foreign animal disease viruses; and the need for confirmatory testing protocol development.

Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).