One-step RT-qPCR Research Articles

First Detection of Hepatitis E Virus RNA in Ovine Raw Milk from Herds in Central Italy.

HEV mainly enters animal and human hosts through the orofecal route, which presents a critical health concern alongside the associated environmental variable. Among products of animal origin, milk (both ovine and bovine) can harbor HEV RNA, which can potentially be transmitted to consumers. In this study, a total of 220 raw ovine milk samples were collected from Apennine breed subjects farmed (transhumance method) in three different Italian provinces, L'Aquila, Pescara, and Teramo, located in the Abruzzo region (Central Italy). All the specimens were screened using one-step real-time RT-qPCR and nested RT-PCR assays. Among them, 5/220 or 2.27% harbored HEV RNA fragments belonging to the ORF1 and ORF2 codifying regions of the genotype 3c. The average viral amount discovered was 102 GE/mL. These subjects represented 2/57 or 3.51% of the Pescara herd, and 3/105 or 2.86% of the Teramo herd. Although HEV RNA was discovered in sheep fecal samples, the original data obtained in the present study represent the first HEV RNA detection in ovine raw milk from Italy.

Molecular characterization of peste des petits ruminants virus and Mycoplasma capricolum subsp. capripneumoniae in small ruminants in northern Mauritania, 2023.

Global eradication of peste des petits ruminants (PPR) is planned for 2030 by international animal health organizations in collaboration with national partners. As the deadline approaches, it is fundamental that the PPR status in each country is determined. In addition, the identification of other pathogens of small ruminants that share common geographical locations and can produce similar clinical signs is also important for differential diagnosis. With this in mind, 37 samples collected from goats and sheep presenting respiratory symptoms in Mauritania in 2023 were screened for the presence of PPR virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies capripneumoniae (Mccp) using a one-step multiplex RT-qPCR assay. None of the samples were positive for Capripoxvirus or P. multocida. Nine of them were positive for PPRV and sequence analysis of a segment of the PPRV nucleoprotein revealed that they belonged to lineage IV and were similar to viruses recently identified in Côte D'Ivoire, Guinea, and Niger indicating transboundary movement. The full genome of one representative virus was also generated. Mccp was identified in eight samples and multi-locus sequence analysis (MLSA) identified them as belonging to MLSA Group 3 together with Mccps identified in China, Tajikistan, Turkey and the United Arab Emirates. This is the first time that such a study has been undertaken in Mauritania and the data generated should be of interest to those involved in the management of goat diseases in Mauritania and neighbouring countries.

Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2

BackgroundThere is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals.MethodsThis study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity.ResultsThe research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen’s kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 101 and 100% specificity.ConclusionsBased on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission.

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.

RNA isolation by two different methods in analysing adipose-biomarkers of mouse adipose tissues

The RNeasy Mini Kit and the TRI Reagent protocol are commonly employed techniques for extracting total RNA and are widely utilised in research pertaining to the expression of adipose biomarkers. This study was undertaken to establish and determine an appropriate method for isolating RNA from frozen adipose tissue, with the objective of analysing adipose biomarkers. Total RNA was extracted from frozen white adipose tissues of mice (-80oC) using both the RNeasy Mini Kit and the TRI Reagent protocol. The concentration and purity of the RNA were assessed using NanoDrop. One-step RT-qPCR was employed to quantify the mRNA expression levels of three adipose biomarkers: MEST, SFRP5, and SCD1. The results of this investigation indicated that the RNeasy Mini Kit yielded a higher RNA concentration when compared to the TRI Reagent method. Furthermore, the RNeasy Mini Kit provided RNA samples with superior purity, as evidenced by a higher 260/230 ratio. Although RNA extracted using the TRI Reagent exhibited slightly elevated mRNA expression levels for the three biomarkers, these differences did not reach statistical significance. In summary, both methods are suitable for total RNA extraction, but the RNeasy Mini Kit is recommended for obtaining higher RNA concentration and purity. The choice of method should be contingent upon the intended downstream application of the RNA.

Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance.

The major arboviruses mainly belong to the Bunyaviridae, Togaviridae, and Flaviviridae families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R2 = 0.99, for DENV-2 and DENV-3; R2 = 0.98, for DENV-4; and CHIKV; R2 = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10-1 copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 101 for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance.

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.

Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates.

Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R2) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples.

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.

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.

Simultaneous detection of human norovirus GI, GII and SARS-CoV-2 by a quantitative one-step triplex RT-qPCR.

There are many similarities in the clinical manifestations of human norovirus and SARS-CoV-2 infections, and nucleic acid detection is the gold standard for diagnosing both diseases. In order to expedite the identification of norovirus and SARS-CoV-2, a quantitative one-step triplex reverse transcription PCR (RT-qPCR) method was designed in this paper. A one-step triplex RT-qPCR assay was developed for simultaneous detection and differentiation of human norovirus GI (NoV-GI), GII (NoV-GII) and SARS-CoV-2 from fecal specimens. The triplex RT-qPCR assay had high detection reproducibility (CV < 1%) and sensitivity. The lower limits of detection (LLOD95) of the triplex RT-qPCR assay for each target site were 128.5-172.8 copies/mL, and LLOD95 of the singleplex RT-qPCR assay were 110.3-142.0 copies/mL. Meanwhile, among the detection of clinical oropharyngeal swabs and fecal specimens, the results of the singleplex and triplex RT-qPCR assay showed high agreement. The triplex RT-qPCR assay for simultaneous detection of NoV-GI, NoV-GII and SARS-CoV-2 from fecal specimens has high clinical application value.

An ultrasensitive and multiplexed miRNA one-step real time RT-qPCR detection system and its application in esophageal cancer serum

MicroRNAs (miRNAs) are increasingly recognized as promising biomarkers for early disease diagnosis and prognosis. Therefore, the need for rapid, robust methods for multiplex miRNA detection in biological research and clinical diagnosis is crucial. This study introduces a novel multiplex miRNA detection method, SMOS-qPCR (Sensitive and Multiplexed One-Step RT-qPCR). The method integrates multiplexed reverse transcription and TaqMan-based qPCR into a single tube, employing a one-step operation on a real-time PCR system. We investigated the effect of 3′ end phosphorylation of the Linker, Linker concentration and probe concentration on the SMOS-qPCR, resulted in a wide linear range from 1 fM to 0.1 zM (R2 ≥ 0.99 for each miRNA), surpassing the capabilities of stem-loop RT-qPCR and SYBR Green One-step RT-qPCR. The method showed excellent performance in distinguishing mature miRNA from miRNA precursor, and successfully detected four miRNAs in a single tube without cross-interference. Its high specificity enables precise differentiation of less than 1% nonspecific signal. Finally, we demonstrated the effectiveness of the SMOS-qPCR system in detecting circulating miRNAs in serum samples, distinguishing between esophageal cancers and health individuals with high AUC values (>0.940). In conclusion, the proposed SMOS-qPCR system offers a straightforward and promising approach for miRNA profiling in future clinical applications.

Rapid and Visual Detection of Actinidia Chlorotic Ringspot-Associated Virus Using One-Step Reverse-Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay.

Actinidia chlorotic ringspot-associated virus (AcCRaV) occurs widely in major kiwifruit producing areas of China and is often accompanied by coinfecting viruses, affecting the growth, yield, and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for diagnosing and developing effective AcCRaV management strategies. In this study, a one-step reverse-transcription recombinase polymerase amplification combined with a lateral flow dipstick (RT-RPA-LFD) assay was developed for rapid detection of AcCRaV. Specific primers and a probe were designed based on the conserved region of the coat protein gene sequence of AcCRaV. The one-step RT-RPA reaction can be performed at 35 and 40°C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limit of the one-step RT-RPA-LFD assay was 10-8 ng (about 20 viral copies), which was equal with one-step RT-qPCR and 100 times more sensitive than one-step RT-PCR. Moreover, the one-step RT-RPA-LFD assay was successfully applied to detect AcCRaV from crude extracts, and the entire detection process can be completed within 40 min. These results indicate that the RT-RPA-LFD assay is a simple, rapid, and sensitive strategy that can be used for accurate diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying the one-step RT-RPA-LFD assay to detect a kiwifruit virus.

TaqMan probe-based one-step multiplex real-time RT-PCR assay for the diagnosis of foot-and-mouth disease

Effective control and monitoring the spread of foot-and-mouth disease (FMD) relies upon rapid and accurate laboratory detection of FMD virus (FMDV). Therefore, in this report, a multiplex TaqMan probe-based one-step RT-qPCR assay simultaneously targeting FMDV 5′UTR and 3Dpol regions, and 18S rRNA housekeeping gene (as an internal control) in a single reaction tube was developed and evaluated. The multiplex one-step RT-qPCR assay specifically detected viral genome in both FMDV-infected cell culture suspensions and clinical samples collected from known-FMD infected animals. The assay could detect FMDV RNA in the archived FMDV cell culture isolates (n = 120) collected during the last two decades in India. In addition, the new assay could also detect viral RNA in the FMD suspected clinical samples (n = 740) collected from various field outbreaks. At a cut-off Ct-value of &amp;lt;38, the assay could detect at least 20 and 10 copies of FMDV 3Dpol and 5′UTR genes, respectively. Further, the multiplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.3% to 3.03% for FMDV-3Dpol gene target, and from 1.44% to 4.69% for 5′UTR gene target. In addition, it was found that the new assay could be used to detect viral genome in a variety of samples (epithelium, saliva, OPF, milk and blood) without any significance difference in the detection limit of the assay. Hence, the multiplex one-step RT-qPCR assay could be considered a valuable tool for the detection of FMDV in India.

Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses

BackgroundDuring the coronavirus disease 2019 (COVID-19) pandemic, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Influenza A, and Influenza B viruses is essential for rapid differential diagnosis in patients with similar symptoms, especially during “flu season” in the post-pandemic era. So far, several multiplex methods have been approved for the simultaneous detection of SARS-CoV-2, Influenza A, and Influenza B. However, due to the rapid mutation rate of the SARS-CoV-2 genome and the emergence of new variants, existing methods must be improved and updated.MethodsTo identify a highly conserved region in the SARS-CoV-2 N-gene, a genomic survey was performed to increase the sensitivity and specificity of primer and probe sets targeting the SARS-CoV-2 genome. The 95% LLOD (95% lower limits of detection) were calculated by probit analysis. A total of 70 predetermined clinical samples using singleplex RT-qPCR assays, were included. The clinical performance of the multiplex RT-qPCR assay was determined and compared with a commercial multiplex kit. The Cohen’s kappa coefficient, P-value (McNemar’s test), Passing-Bablok regression, and Bland Altman agreement analysis were determined to monitor the agreement of the assays.ResultsThe novel SARS-CoV-2 primer and probe set designed in this assay was able to detect all variants of concern (VOCs) and variants of interest (VOIs) with high analytical and clinical performance. The 95% LLOD for the multiplex RT-qPCR was 20 copies per reaction for the N gene of SARS-CoV-2, 2 copies per reaction for M1 gene of Influenza A and NS1 gene of Influenza B. The diagnostic sensitivity of the multiplex RT-qPCR was 94.4%, 93.7%, and 100% for the detection of SARS-CoV-2, Influenza A, and Influenza B genomes, respectively. Moreover, the specificity was identical (100%) in both assays. According to the agreement analysis results, there was no statistical difference between our multiplex assay and the commercial kit.ConclusionsIn this study, we developed a novel in-house made multiplex RT-qPCR assay, with high sensitivity, specificity, and reliability for the diagnosis of SARS-CoV-2 infection in clinical samples. This is valuable during Influenza seasons when influenza co-circulates with SARS-CoV-2, as it saves costs, time, and thus specific and timely treatment of patients.

Evidence for the circulation of genotype 4 Eurasian avian-like lineage swine H1N1 influenza A viruses on Korean swine farms, obtained using a newly developed one-step multiplex RT-qPCR assay.

Genotype 4 (G4) Eurasian avian-like lineage swine H1N1 influenza A viruses, which are reassortants containing sequences from the pandemic 2009 H1N1 virus lineage, triple-reassortant-lineage internal genes, and EA-lineage external genes, have been reported in China since 2013. These have been predominant in pig populations since 2016 and have exhibited pandemic potential. In this study, we developed a one-step multiplex RT-qPCR assay targeting the M, HA1, and PB2 genes to detect G4 and related EA H1N1 viruses, with detection limits of 1.5 × 101 copies/μL and 1.15 × 10-2 ng/μL for the purified PCR products and RNA templates, respectively. The specificity of the detection method was confirmed using various influenza virus subtypes. When the one-step multiplex RT-qPCR assay was applied to swine respiratory samples collected between 2020 and 2022 in Korea, a virus related to G4 EA H1N1 strains was detected. Phylogenetic analysis based on portions of all eight genome segments showed that the positive sample contained HA, NA, PB2, NS, and NP genes closely related to those of G4 EA H1N1 viruses, confirming the ability of our assay to accurately detect G4 EA H1N1 viruses in the field.

Trends in SARS-CoV-2 clinically confirmed cases and viral load in wastewater: A critical alignment for Padua city (NE Italy)

Since the beginning of the COVID-19 pandemic, wastewater-based epidemiology (WBE) has been depicted as a promising environmental surveillance tool and early warning system. Predictive models for the estimate of COVID-19 cases from wastewater viral loads also earned lot of interest and are currently under development. Hereby a pilot study that compares WBE surveillance data with confirmed cases, total hospitalizations, doses of vaccine administered and predominance of coronavirus variants.Composite 24hrs wastewater samples were collected weekly between September 2021 and July 2022 from Padua wastewater treatment plant. Samples were processed following a previously published method. One-step RT-qPCR was performed for quantification, adapting an Orf1b-nsp14 gene assay. Variant replacement was derived from the monthly bulletins of the Italian National Health Institute. Aggregate data on vaccine doses administered and on COVID-19 prevalence and hospitalizations were retrieved from official reports.Eighty-two samples were processed. Viral loads highlighted 3 major peaks in January, April and July 2022. Quantitation of SARS-CoV-2 in wastewater and clinical surveillance resulted temporally juxtaposable. However, variation of the two curves is not proportional. SARS-CoV-2 showed its highest peak in April, whereas maximum COVID-19 prevalence was achieved in January. Total hospitalizations followed the prevalence trend. Omicron BA.1 started to replace the Delta variant in December 2021. Subsequently, the shift towards Omicron BA.2 occurred between February and April 2022. Finally, BA.4/5 attested around June, somehow preceding the summer peak.Emergence of Omicron BA.1 over Delta could be a possible driver of the increase in both clinical cases and wastewater viral load in January 2022. In late March 2022, Omicron BA.2 replaced BA.1: this reflected in a steep increase of wastewater viral load, but not of COVID-19 confirmed cases. When a dramatic drop in the testing capacity of clinical surveillance occurred, WBE was possibly capable of detecting a substantial increase in viral circulation.

A-304 A RT-PCR Assay for Detection and Quantification of Hepatitis D Virus (HDV)

Abstract Background Hepatitis D virus (HDV), also called Delta Hepatitis, is associated with the presence of the Hepatitis B virus (HBV) and is responsible for the infection and inflammation of liver cells. It is estimated that 15–20 million people worldwide are affected by HDV. In Brazil, Hepatitis D has high prevalence rates in the Amazon Basin. HDV is an enveloped virus with a 36 nm average diameter. The HDV virion involves a negative-sense, single-stranded circular RNA molecule associated with an inner-core delta antigen surrounded by envelope glycoproteins. HDV-infected individuals may not show signs or symptoms of the disease. When present, the most common are: tiredness, dizziness, nausea and/or vomiting, fever, abdominal pain, and yellow skin/eyes. Chronic Hepatitis D is considered the most severe form of chronic viral hepatitis, with more rapid progression to cirrhosis and an increased risk for decompensation, hepatocellular carcinoma (HCC), and death. Molecularly, determining the concentration of virus circulating in the blood of HDV patients is extremely important for diagnosis. The Quantitative Polymerase Chain Reaction (qPCR) is a molecular technique capable of detecting the viral genome with high sensitivity and specificity, ensuring the safety of the result. Therefore, this study aims to describe the analytical validation of a one-step RT-qPCR assay for HDV detection. Methods Assay performance was evaluated using commercial quantified positive control to HDV (1000, 100, 10, 1, and 0.5 IU/µL), and the parameters of analysis included: standardization assays, efficiency, and detection limit. For specificity evaluation, positive samples for Epstein-Barr Virus (EBV), Herpes Simplex Virus (HSV), Cytomegalovirus (CMV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) were used. RNA isolation was performed with the MagNA Pure 24 Total NA Isolation Kit at the automated platform MagNA Pure 24 System (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions. Altona RealStar HDV RT-PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) was used to directly amplify RNA samples on the qPCR instrument, according to the manufacturer's instructions. Results The standardization assays indicated good qPCR amplification curves and high fluorescence levels between the controls evaluated. The assay demonstrated an average efficiency of 95%, with a coefficient of determination (r2) of 0.99. The detection limit of the test was 1 IU/µL for the HDV target (95% confidence interval). No cross-reactions with closely related viruses were observed (15 HDV-negative human plasma samples), indicating 100% specificity. The experiments performed to evaluate the precision demonstrated optimal repeatability and reproducibility. Conclusion The HDV diagnostic is required to ensure sensitive, specific and accurate identification of the viral agent and thus prompt surveillance actions such as epidemiological monitoring and control measures of infection. The RT-qPCR assay described here provides a reliable, highly specific, and sensitive method for the detection and quantification of HDV. Nevertheless, the assay needs to be evaluated in conjunction with a clinical and epidemiological context.

Development and validation of a duplex RT-qPCR assay for norovirus quantification in wastewater samples

Norovirus (NoV) is a highly contagious enteric virus that causes widespread outbreaks and a substantial number of deaths across communities. As clinical surveillance is often insufficient, wastewater-based epidemiology (WBE) may provide novel pathways of tracking outbreaks. To utilise WBE, it is important to use accurate and sensitive methods for viral quantification. In this study, we developed a one-step duplex RT-qPCR assay to simultaneously test the two main human pathogenic NoV genogroups, GI and GII, in wastewater samples. The assay had low limits of detection (LOD), namely 0.52 genome copies (gc)/µl for NoVGI and 1.37 gc/µl for NoVGII. No significant concentration-dependent interactions were noted for both NoVGI and for NoVGII when the two targets were mixed at different concentrations in the samples. When tested on wastewater-derived RNA eluents, no significant difference between duplex and singleplex concentrations were found for either target. Low levels of inhibition (up to 32 %) were noted due to organic matter present in the wastewater extracts. From these results we argue that the duplex RT-qPCR assay developed enables the sensitive detection of both NoVGI and NoVGII in wastewater-derived RNA eluents, in a time and cost-effective way and may be used for surveillance to monitor public and environmental health.