RT-qPCR Protocol Research Articles

First Report on the Molecular Detection of Canine Astrovirus (CaAstV) in Dogs with Gastrointestinal Disease in Ecuador Using a Fast and Sensitive RT-qPCR Assay Based on SYBR Green®.

Enteric viruses are responsible for a significant number of gastrointestinal illnesses in dogs globally. One of the main enteric viruses is the canine astrovirus (CaAstV), which causes diarrhea in dogs of various ages. It is linked to symptoms such as diarrhea, vomiting, depression and a significant mortality rate due to gastrointestinal disorders. It is a single-stranded positive RNA virus, with three open reading frames, ORF1a, ORF1b and ORF2, where the last one codes for the virus capsid protein and is the most variable and antigenic region of the virus. The aim of this work is to develop and standardize a quick detection method to enable the diagnosis of this etiological agent in dogs with gastroenteritis in Ecuador in order to provide prompt and suitable treatment. The assay was specific for amplification of the genome of CaAstV, as no amplification was shown for other canine enteric viruses (CPV-2, CCoV and CDV), sensitive by being able to detect up to one copy of viral genetic material, and repeatable with inter- and intra-assay coefficients of variation of less than 10% between assays. The standard curve showed an efficiency of 103.9%. For the validation of this method, 221 fecal samples from dogs affected with gastroenteritis of various ages from different provinces of Ecuador were used. From the RT-qPCR protocol, 119 samples were found positive for CaAstV, equivalent to 53.8% of the samples processed. CaAstV was detected in dogs where both the highest virus prevalence in the tested strains and the highest viral loads were seen in the younger canine groups up to 48 weeks; in addition, different strains of the virus were identified based on a sequenced fragment of ORF1b, demonstrating the first report of the presence of CaAstV circulating in the domestic canine population affected by gastroenteritis in Ecuador, which could be associated with the etiology and severity of enteric disease.

The potential of tailed amplicons for SARS-CoV-2 detection in Nucleic Acid Lateral Flow Assays.

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/μL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.

Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR.

Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.

FlashPCR: Revolutionising qPCR by Accelerating Amplification through Low ∆T Protocols.

Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called "FlashPCR", uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications.

Detection of Chikungunya Virus RNA in Oral Fluid and Urine: An Alternative Approach to Diagnosis?

To evaluate whether oral fluids (OF) and urine can serve as alternative, non-invasive samples to diagnose chikungunya virus (CHIKV) infection via RT-qPCR, we employed the same RNA extraction and RT-qPCR protocols on paired serum, OF and urine samples collected from 51 patients with chikungunya during the acute phase of the illness. Chikungunya patients were confirmed through RT-qPCR in acute-phase sera (N = 19), IgM seroconversion between acute- and convalescent-phase sera (N = 12), or IgM detection in acute-phase sera (N = 20). The controls included paired serum, OF and urine samples from patients with non-arbovirus acute febrile illness (N = 28) and RT-PCR-confirmed dengue (N = 16). Nine (47%) of the patients with positive RT-qPCR for CHIKV in sera and two (17%) of those with CHIKV infection confirmed solely via IgM seroconversion had OF positive for CHIKV in RT-qPCR. One (5%) patient with CHIKV infection confirmed via serum RT-qPCR was positive in the RT-qPCR performed on urine. None of the negative control group samples were positive. Although OF may serve as an alternative sample for diagnosing acute chikungunya in specific settings, a negative result cannot rule out an infection. Further research is needed to investigate whether OF and urine collected later in the disease course when serum becomes RT-qPCR-negative may be helpful in CHIKV diagnosis and surveillance, as well as to determine whether urine and OF pose any risk of CHIKV transmission.

An Open One-Step RT-qPCR for SARS-CoV-2 detection.

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases

Circulating miRNAs have potential as minimally invasive biomarkers for diagnosing various diseases, including ageing-related disorders such as Alzheimer’s disease (AD). However, the lack of standardization in the common analysis method, RT-qPCR, and specifically in the normalization step, has resulted in inconsistent data across studies, hindering miRNA clinical implementation as well as basic research. To address this issue, this study proposes an optimized protocol for key steps in miRNA profiling, which incorporates absorbance-based haemolysis detection for assessing sample quality, double spike-in controls for miRNA isolation and reverse transcription, and the use of 7 stable normalizers verified in an aging population, including healthy subjects and individuals at different stages of Alzheimer's disease (140 subjects). The stability of these 7 normalizers was demonstrated using our novel method called BestmiRNorm for identifying optimal normalizers. BestmiRNorm, developed utilizing the Python programming language, enables the assessment of up to 11 potential normalizers. The standardized application of this optimized RT-qPCR protocol and the recommended normalizers are crucial for the development of miRNAs as biomarkers for AD and other ageing-related diseases in clinical diagnostics and basic research.

Rapid response of a public health reference laboratory to the COVID-19 pandemic.

Introduction. Brazil was one of the most affected countries by the COVID-19 pandemic. Instituto Adolfo Lutz (IAL) is the reference laboratory for COVID-19 in São Paulo, the most populous state in Brazil. In April 2020, a secondary diagnostic pole named IAL-2 was created to enhance IAL's capacity for COVID-19 diagnosis.Hypothesis/Gap Statement. Public health laboratories must be prepared to rapidly respond to emerging epidemics or pandemics.Aim. To describe the design of IAL-2 and correlate the results of RT-qPCR tests for COVID-19 with secondary data on suspected cases of SARS-CoV-2 infection in the São Paulo state.Methodology. This is a retrospective study based on the analysis of secondary data from patients suspected of infection by SARS-CoV-2 whose clinical samples were submitted to real-time PCR after reverse transcription (RT-qPCR) at IAL-2, between 1 April 2020 and 8 March 2022. RT-qPCR Ct results of the different kits used were also analysed.Results. IAL-2 was implemented in April 2020, just over a month after the detection of the first COVID-19 case in Brazil. The laboratory performed 304,250 RT-qPCR tests during the study period, of which 98 319 (32.3 %) were positive, 205827 (67.7 %) negative, and 104 (0.03 %) inconclusive for SARS-CoV-2. RT-qPCR Ct values≤30 for E/N genes of SARS-CoV-2 were presented by 79.7 % of all the samples included in the study.Conclusion. IAL was able to rapidly implement a new laboratory structure to support the processing of an enormous number of samples for diagnosis of COVID-19, outlining strategies to carry out work with quality, using different RT-qPCR protocols.

Evaluation of SYBR Green Based Real-Time Polymerase Chain Reaction (qPCR) in Detection of Reovirus

Avian reovirus (ARV) belongs to the Orthoreovirus genus and Reoviridae family and contains icosahedral symmetry with a double-shelled arrangement of surface protein. Avian reoviruses infection can often be seen in a broad range of avian species, including chickens, ducks, pigeons, and turkeys. These viruses can cause tenosynovitis, causing acute lameness due to swelling of one or both hock joints, and can be associated with malabsorption, hepatitis, myocarditis, respiratory disease, and enteritis. This study objective was to use the BRYT Green® based real-time RT-PCR assay in targeting the S4 segment gene for the detection of AVR, thus improving the efficiency and cost of the diagnosis. Thirty-one (n=31), including known ARV-positive samples consisting of pool organs, liver, tendon, and allantoic fluid, were selected to be tested using BRYT Green®-based real-time RT-PCR. The finding of this study showed the one-step BRYT Green®-based RT-qPCR protocol for the detection of the Reovirus S4 genome segment is a success and has advantages compared with conventional RT-PCR. This assay is time-efficient, cost-effective, user-friendly, sensitive, and highly specific. There is no doubt that this protocol can be used in many laboratory diagnoses for avian reovirus infection.

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area.

The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by official organizations. Among those, real-time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for the diagnosis and quantification of SJ and RG type VNNV using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10- to 100-fold lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8-6.8 × 104 (SJ type) or 1.04 × 101-1.04 × 105 (RG type) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV.

Development and validation of a new quantitative reverse transcription PCR assay for the diagnosis of human sporotrichosis.

Sporotrichosis is an emergent public health problem. The mycological diagnosis of this infection is based on culture, which is fastidious and may represent a biohazard for technicians. Although not widely implemented in routine diagnosis, molecular methodologies are fast, have good accuracy, and can be easily standardized, aiding in the early diagnosis of neglected mycoses. This study aimed at implementing a new pan-Sporothrix quantitative reverse transcription PCR (RT-qPCR) assay, and then validating it on clinical samples from confirmed human sporotrichosis cases. Sixty-eight human samples with culture-confirmed diagnostic of sporotrichosis were collected from 64 patients followed at a Brazilian reference center for endemic mycoses. These samples were submitted to whole nucleic acids extraction, followed by a RT-qPCR protocol. The limit of detection was 244 fg, the efficiency was 2.0 (100%), and the assay could amplify the genetic material of the three major clinically relevant species of the genus Sporothrix. Among the 68 samples analyzed, 62 were positive in RT-qPCR, showing an overall sensitivity of 91.18%, which variated according to the type of biological sample: 96.72% in skin samples (n=61), 100% in respiratory (n=3), whereas all cerebrospinal fluid specimens (n=4) were negative. The specificity was 100% when tested in 25 samples from patients with other mycoses and tuberculosis. In addition, DNA from 93 fungal species did not yield positive results, confirming the high specificity of this test. Our RT-qPCR presented high sensitivity and specificity, representing an excellent tool for a fast and reliable diagnosis of human sporotrichosis.

Method versatility in RNA extraction-free PCR detection of SARS-CoV-2 in saliva samples

Early in the pandemic, a simple, open-source, RNA extraction-free RT-qPCR protocol for SARS-CoV-2 detection in saliva was developed and made widely available. This simplified approach (SalivaDirect) requires only sample treatment with proteinase K prior to PCR testing. However, feedback from clinical laboratories highlighted a need for a flexible workflow that can be seamlessly integrated into their current health and safety requirements for the receiving and handling of potentially infectious samples. To address these varying needs, we explored additional pre-PCR workflows. We built upon the original SalivaDirect workflow to include an initial incubation step (95 °C for 30 min, 95 °C for 5 min or 65 °C for 15 min) with or without addition of proteinase K. The limit of detection for the workflows tested did not significantly differ from that of the original SalivaDirect workflow. When tested on de-identified saliva samples from confirmed COVID-19 individuals, these workflows also produced comparable virus detection and assay sensitivities, as determined by RT-qPCR analysis. Exclusion of proteinase K did not negatively affect the sensitivity of the assay. The addition of multiple heat pretreatment options to the SalivaDirect protocol increases the accessibility of this cost-effective SARS-CoV-2 test as it gives diagnostic laboratories the flexibility to implement the workflow which best suits their safety protocols.

Comparison of diagnostic performance of RT-qPCR, RT-LAMP and IgM/IgG rapid tests for detection of SARS-CoV-2 among healthcare workers in Brazil

BackgroundCOVID-19 has become a major public health problem after the outbreak caused by SARS-CoV-2 virus. Great efforts to contain COVID-19 transmission have been applied worldwide. In this context, accurate and fast diagnosis is essential. MethodsIn this prospective study, we evaluated the clinical performance of three different RNA-based molecular tests – RT-qPCR (Charité protocol), RT-qPCR (CDC (USA) protocol) and RT-LAMP – and one rapid test for detecting anti-SARS-CoV-2 IgM and IgG antibodies. ResultsOur results demonstrate that RT-qPCR using the CDC (USA) protocol is the most accurate diagnostic test among those evaluated, while oro-nasopharyngeal swabs are the most appropriate biological sample. RT-LAMP was the RNA-based molecular test with lowest sensitivity while the serological test presented the lowest sensitivity among all evaluated tests, indicating that the latter test is not a good predictor of disease in the first days after symptoms onset. Additionally, we observed higher viral load in individuals who reported more than 3 symptoms at the baseline. Nevertheless, viral load had not impacted the probability of testing positive for SARS-CoV-2. ConclusionOur data indicates that RT-qPCR using the CDC (USA) protocol in oro-nasopharyngeal swabs samples should be the method of choice to diagnosis COVID-19.

RT-qPCR Protocol Optimization Assessment with Propidium Monoazide for Viable and Non-Viable Pathogen Detection

Real time PCR or RT-qPCR (including reverse transcriptase PCR) is a gold standard in diagnostic testing for pathogens such as bacteria and viruses that contain RNA or DNA for amplification. As a validated molecular assay technique, RT- qPCR has been instrumental in microbial detection because of its high throughput, quantification of DNA or RNA samples as well as quick turnaround especially when compared with culturing methods that can take multiple days to generate result. While the advancement of PCR has had significant impact for diagnosis of diseases such as COVID- 19through its high sensitivity and specificity, the test also brings with it its own challenges of false positive and false negative results. This is normally attributed to PCR's susceptibility to contamination or inhibitors. However, there are other conditions like DNA extraction techniques and protocols in PCR methodology that can impact the accuracy of the PCR results as well. This paper, therefore, aims to synthesize factors that contribute to false positive and negatives in PCR results, improvements to protocols that includes the use of DNA intercalating dyes such as Propidium Monoazide (PMA) in PCR assay to improve DNA extraction and discuss the importance of an optimized PCR assay for diagnostic testing efficacy from a social and economic standpoint.

A new protocol for validation of Chondro, Adipo and Osteo differentiation kit of Cultured Adipose-Derived Stem Cells (ADSC) by real-time rt-QPCR

Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose derived Stem Cells) show a great potential for degenerative diseases treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validated a commercially available kit able to explore changes in gene expression under osteogenic, adipogenic and chondrogenic differentiation of human ADSC. Initially, we selected a better indicators of trilineage differentiation by using third passages of cultured ADSC from stromal vascular fraction (SVF) isolated from fresh adipose tissue by enzymatic digestion. On the basis of statistically significant results ACAN, FABP4A and Col11a1 were chosen as indicators of chondrogenic, adipogenic and osteogenic differentiation respectively. An in-vitro aging analysis was then performed to evaluate the ADSC passage with the highest differentiation potential. Total RNA extraction from induced differentiation and controls ADSC from passage 2–6 and relative quantifications of mRNA expression of selected genes were performed according to rt-PCR kits tested. The chondrogenic differentiation test showed equivalent ∆∆Ct values for ACAN detection for cell passages ranging from P3 to P6, proving that they can be considered as equivalent samples for differentiation assays evaluation. For what concerns adipogenic differentiation and FABP4 detection, similar results were observed in all the cell passages tested; on the contrary only passage P6 showed suitable ∆∆Ct values for Col11a1 detection for osteogenic differentiation evaluation. In conclusion, we have validated a suitable real-time rt-QPCR protocol for osteogenic, chondrogenic and adipogenic ADSC differentiation ability evaluation in-vitro.

Novel duplex RT-qPCR for animal rabies surveillance.

Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence (DI) followed by mouse inoculation test (MIT). WHO recommends molecular biology techniques such as RT-qPCR for replacing MIT to diagnose rabies in animal samples. Recently, a real-time PCR protocol that detects all rabies virus variants identified worldwide was validated. This assay is a pan-Lyssavirus TaqMan quantitative RT-PCR called LN34. A modified LN34 assay protocol was tested at the Paraná State Reference Laboratory (Lacen/PR) using animal samples previously tested by DI and MIT, the gold standard (GS). This method has been changed to a RT-qPCR duplex format to better fit the diagnostic routine. The new assay was called duplex LN34 and β-actin RT-qPCR. All the 88 samples evaluated using the GS test, modified pan-Lyssavirus TaqMan RT-qPCR and duplex LN34 and β-actin RT-qPCR showed 100% agreement with each other. This novel duplex RT-qPCR protocol has shown adequate diagnostic performance and may be used in research and surveillance purposes, replacing the standard MIT and ending mice use for rabies diagnosis.

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Background: Recent estimates indicate that the COVID-19 pandemic, which is caused by the SARS-CoV-2 virus, could be effectively controlled via the development and implementation of diagnostic tools such as quantitative reverse transcription PCR (RT-qPCR). However, this reaction often generates false-negative results due to novel mutations and can also be affected by the secondary structure of the RNA transcripts that derive from the gene sequence used for diagnostic purposes. Methods: Using high-performance computing, we consolidated a global SARS-CoV-2 genome repository encompassing 19,317 genomes from the GenBank database and 107,259 from the GISAID database to generate monthly SARS-CoV-2 consensus sequences from January to December 2020. Results: These sequences were then used to create ORF8-specific primers and probes to validate single and multiplex RT-qPCR protocols both in silico and experimentally using genes E (Berlin protocol) and N (CDC protocol) as targets. Conclusions: Our findings demonstrated that RT-qPCR Ct values were improved by the inclusion of either a denaturing solution composed of tetraethylammonium chloride (TEA) and dimethyl sulfoxide (DMSO) and by adjusting nucleotide proportions based on the SARS-CoV-2 genome.

RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step.

Reverse transcription of RNA coupled to amplification of the resulting cDNA by the polymerase chain reaction (RT-PCR) is one of the principal molecular technologies in use today, with applications across all areas of science and medicine. In its real-time, fluorescence-based usage (RT-qPCR), it has long been a core technology driving the accurate, rapid and sensitive laboratory diagnosis of infectious diseases. However, RT-qPCR protocols have changed little over the past 30 years, with the RT step constituting a significant percentage of the time taken to complete a typical RT-qPCR assay. When applied to research investigations, reverse transcription has been evaluated by criteria such as maximum yield, length of transcription, fidelity, and faithful representation of an RNA pool. Crucially, however, these are of less relevance in a diagnostic RT-PCR test, where speed and sensitivity are the prime RT imperatives, with specificity contributed by the PCR component. We propose a paradigm shift that omits the requirement for a separate high-temperature RT step at the beginning of an RT-qPCR assay. This is achieved by means of an innovative protocol that incorporates suitable reagents with a revised primer and amplicon design and we demonstrate a proof of principle that incorporates the RT step as part of the PCR assay setup at room temperature. Use of this modification as part of a diagnostic assay will of course require additional characterisation, validation and optimisation of the PCR step. Combining this revision with our previous development of fast qPCR protocols allows completion of a 40 cycle RT-qPCR run on a suitable commercial instrument in approximately 15 min. Even faster times, in combination with extreme PCR procedures, can be achieved.

A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes.

Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x104 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100. In full combination assays, CHIKV was detected to 1.32x104, DENV1-4 to 3.79x101 and ZIKV to 1.07x100. This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization

More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.