Commercial Real-time PCR Research Articles

Molecular diagnosis of neglected infectious agents of heep and attle abortions: the prevalences of Coxiella burnetii, Francisella tularensis and Chlamydophila abortus at a glance

Bacterial abortive agents such as Brucella spp., Salmonella spp., Campylobacter spp., Listeria spp. cause serious infections that lead to significant economic losses in cattle and sheep breeding. These bacteria can be easily cultured under in vitro laboratory conditions. Abortions caused by intracellular bacteria such as Coxiella burnetii (C. burnetii), Chlamydophila abortus (C. abortus) and Francisella tularensis (F. tularensis) are less prevalent in abortive cases and the diagnosis of these bacteria, many of which need to cell culture for cultivation and biosafety level-3 laboratory facilities for safe working, can be made by PCR. In this study, it was aimed to determine the molecular prevalence of C. burnetii, C. abortus and F. tularensis agents, which were neglected during the diagnosis of abortions, in cattle and sheep collected from different regions of Türkiye. A total of 395 clinical materials were analyzed via agent-specific commercial Real-Time PCR. As a result, the molecular prevalence of F. tularensis, C. burnetii and C. abortus was determined as 14%, 2.9% and 2.28%, respectively. F. tularensis in cattle clinical samples and C. burnetii in sheep clinical samples were not found. This is the first report that F. tularensis was found as an agent in an abortive material in Türkiye. Although it differs in terms of prevalence, it has been determined that these microorganisms, which are neglected in routine diagnosis, can be spread with aborted materials, especially vaginal discharge, which may pose a risk of transmission.

Combined Locked Nucleic Acid Probes and High-Resolution Melting Curve Analysis for Detection of Rifampicin-Resistant Tuberculosis in Northern Thailand.

Rifampicin-resistant tuberculosis (RR-TB) has become a major threat globally. This study aims to develop a new assay, RIF-RDp, to enhance the detection of RR-TB based on combined locked nucleic acid (LNA) probes with high-resolution melting curve analysis (HRM). Two new LNA probes were designed to target the class-III and IV mutations of rpoB, H526D, and D516V. LNA probes showed 100% specificity in the detection of mutant targets among characterized and blinded Mycobacterium tuberculosis (Mtb) isolates. The performance of RIF-RDp was evaluated using 110 blinded clinical Mtb isolates in northern Thailand against drug-susceptibility testing (DST), DNA sequencing, and a commercial real-time PCR kit. This assay showed sensitivity and specificity of 94.55% and 98.18% compared to DST, and 96.36% and 100% compared to DNA sequencing. The efficacy of RIF-RDp was comparable to the commercial kit and DNA sequencing. The Cohen’s Kappa statistic showed almost perfect agreement between RIF-RDp and the commercial kit (κ = 0.95), and RIF-RDp and DNA sequencing (κ = 0.96). Furthermore, this is the first report of the rare mutation profiles, S531W, and a triple codon deletion (510–512) in northern Thailand. According to high accuracy, the RIF-RDp assay may render an easy-to-use, low-cost, and promising diagnostics of RR-TB in the future.

A miniaturized and integrated dual-channel fluorescence module for multiplex real-time PCR in the portable nucleic acid detection system.

A portable nucleic acid detection (PNAD) system based on real-time polymerase chain reaction (real-time PCR) has been developed for point-of-care testing (POCT) of infectious disease pathogens. In order to achieve “sample-in, result-out” while keeping the system compact, the hardware system integrates optical, thermal and motion control modules in a limited space for nucleic acid extraction, purification, amplification and detection. Among these hardware modules, the fluorescence module is one of the most important modules, because its performance directly affects the accuracy and sensitivity of the testing results. In this paper, a miniaturized, high-sensitivity and integrated dual-channel fluorescence module have been proposed for the homemade PNAD system. Based on the principle of confocal optical path, two group of excitation-emission optical paths of different wavelengths are integrated in a small space. In terms of circuitry, a current-light dual negative feedback light emitting diode (LED) drive circuit is applied to improve the stability of the excited light source. All optical and electronic components are integrated in a metal box of 55 mm × 45 mm × 15 mm, that helps miniaturize the detection system. Two different modules have been assembled to fit various fluorescent dyes or probes with the set of excitation and emission as follow: module 1#: 470 nm/525 nm, 570 nm/630 nm; module 2#: 520 nm/570 nm, 630 nm/690 nm. Finally, hepatitis B virus (HBV) concentration gradient detection and multiplex detection of different gene targets of SARS-CoV-2 are carried out on the PNAD system equipped with these two fluorescence modules for evaluating their performances. Compared with the commercial real-time PCR instrument, our fluorescence module has good stability and detection sensitivity.

Development and Characterization of Synthetic Norovirus RNA for Use in Molecular Detection Methods.

Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials. Norovirus GI and GII RNA sequences including the ORF1-ORF2 junction region were designed based on 1,495 reported norovirus sequences and synthesized via plasmid preparation and in vitro transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial norovirus detection kits used in Korea and subjected to homogeneity and stability analyses. A multicenter study involving five laboratories and using four commercial real-time PCR norovirus detection assays was conducted for synthetic norovirus RNA characterization and uncertainty measurements. The synthetic norovirus GI and GII RNAs were positively detected using the six commercial norovirus detection kits and were homogeneous and stable for one year when stored at -20°C or -70°C. All data from the five laboratories were within a range of 1.0 log copies/μL difference for each RNA, and the overall mean concentrations for norovirus GI and GII RNAs were 7.90 log copies/μL and 6.96 log copies/μL, respectively. The synthetic norovirus GI and GII RNAs are adequate for quality control based on commercial molecular detection reagents for noroviruses with high sequence variability. The synthetic RNAs can be used as reference materials in norovirus molecular detection methods.

Detection of Feline Coronavirus Variants in Cats without Feline Infectious Peritonitis

(1) Background: This study aimed to detect feline coronavirus (FCoV) and characterize spike (S) gene mutation profiles in cats suffering from diseases other than feline infectious peritonitis (FIP) using commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) and reevaluating results by sequencing. (2) Methods: In 87 cats in which FIP was excluded by histopathology and immunohistochemistry, FCoV 7b gene and S gene mutation RT-qPCR was performed prospectively on incisional biopsies and fine-needle aspirates of different organs, body fluids, and feces. Samples positive for S gene mutations or mixed FCoV underwent sequencing. (3) Results: In 21/87 cats, FCoV RNA was detectable. S gene mutations were detected by commercial RT-qPCR (and a diagnostic algorithm that was used at the time of sample submission) in at least one sample in 14/21 cats (66.7%), with only mutated FCoV in 2/21, only mixed in 1/21, and different results in 11/21 cats; in the remaining 7/21 cats, RNA load was too low to differentiate. However, sequencing of 8 tissue samples and 8 fecal samples of 9 cats did not confirm mutated FCoV in any of the FCoV RNA-positive cats without FIP. (4) Conclusions: Sequencing results did not confirm results of the commercial S gene mutation RT-qPCR.

Universal Newborn Screening for Congenital Cytomegalovirus Infection - From Infant to Maternal Infection: A Prospective Multicenter Study.

Introduction:Most infants at risk for cytomegalovirus (CMV)-associated sensorineural hearing loss (SNHL) are unrecognized because of the absence of a universal neonatal CMV screening. The search of CMV-DNA by molecular methods in salivary swabs was demonstrated to be a reliable approach. This study describes the results obtained by carrying out a universal screening for congenital CMV (cCMV) infection including all live-born newborns in three Italian sites, as well as the therapeutic interventions and clinical outcome of the CMV-infected neonates. Moreover, CMV maternal infection's characteristics were evaluated.MethodsTo confirm or exclude cCMV infection, a CMV-DNA-positive result on a first salivary swab was followed by repeated saliva and urine samples collected within 21 days of age. Breast milk samples were also collected. The search of CMV-DNA was performed with a single automated quantitative commercial real-time PCR assay, regardless of the type of samples used.ResultsA total of 3,151 newborns were enrolled; 21 (0.66%) of them were congenitally infected (median saliva viral load at screening, 6.65 [range, 5.03–7.17] log10 IU/ml). Very low/low viral load in screening saliva samples (median value, 1.87 [range, 1.14–2.59] log10 IU/ml) was associated with false-positive results (n = 54; 1.7%). CMV-DNA was detected in almost half of the breast milk samples of mother–infant pairs with a false-positive result, suggesting that contamination from breast milk may not be the only explanation in the study population. cCMV infection confirmation with the search of CMV-DNA in a urine sample proved to be the gold standard strategy, since false-positive results were observed in 4/54 (7.5%) of the repeated saliva samples. Symptomatic cCMV infection was observed in 3/21 (14.3%) infants; notably, one (4.7%) developed moderate unilateral SNHL at 5 months after birth. Finally, two symptomatic cCMV infections were associated with primary maternal infection acquired in the first trimester of gestation; one newborn with severe cCMV symptoms was born to a mother with no CMV checkups in pregnancy.ConclusionWithout universal neonatal CMV screening, some infected infants who develop late neurological sequelae may not be recognized and, consequently, they are not able to benefit early from instrumental and therapeutic interventions to limit and/or treat CMV disease.

An HFman probe-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for HIV-1 detection

Loop-mediated isothermal amplification (LAMP) is suitable for the development of a rapid and cost-effective nucleic acid technique for point of care (POC) applications. However, LAMP methods often generate non-specific amplification, therefore inevitably resulting in false positive results especially when sequence-independent dyes are used to indirectly reflect the results. In this study, we established and optimized a reverse transcription LAMP (RT-LAMP) assay with a high-fidelity DNA polymerase-mediated fluorescent probe (HFman probe) for human immunodeficiency virus-1 (HIV-1) detection. The assay showed high sensitivity and specificity. Using 101 plasma samples with different HIV-1 viral load, we demonstrated that our assay can detect the major HIV-1 subtypes circulating in China, including CRF01_AE, CRF07_BC, CRF08_BC, CRF55_01B, and unique recombinant forms (URFs). We also compared our assay with an approved commercial real-time quantitative polymerase chain reaction (RT-qPCR) kit and found the sensitivity, specificity and consistency was 88.8%, 100% and 89.1%, respectively. The HFman probe-based RT-LAMP assay is a high specific detection method that is rapid, variant-tolerant and simple to operate, and thus is of great significance for timely disclosure of HIV status and rapid POC diagnosis.

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay

Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein–protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection.Graphical abstract

Family cluster of Chagas disease among Bolivian immigrants in Italy: High rate of maternal-fetal transmission

BackgroundChagas disease (CD) or American trypanosomiasis is a neglected anthropozoonosis caused by Trypanosoma cruzi that affects 6–8 million people worldwide (mainly in Latin America), 30–40% of whom develop cardiac or digestive complications. Once confined to endemic areas of Latin America, CD has more recently become a global disease as a result of migration flows from endemic to non-endemic regions, particularly in northern America and Europe. Congenital transmission is a particular challenge as it may be sustained for multiple generations and perpetuate the infection even in non-endemic countries. MethodsSubjects were identified during a cross-sectional survey of CD among Latin American people living in Milan, Italy. Serology was carried out using tests based on either a lysate and a recombinant antigen of Trypanosoma cruzi. They were also tested by a conventional Polymerase Chain Reaction (PCR) targeting the 330 bp variable region of the T. cruzi kinetoplast minicircle genome and a commercial real-time PCR. ResultsWe here describe a Bolivian family cluster with seven affected people with at least two autochthonous congenital T. cruzi infection which was identified during the course of a CD screening programme. We also review the epidemiology, diagnosis and control of congenital CD, with particular emphasis on the challenges facing the control and management of such a complex and still largely hidden disease. ConclusionsOur experience confirms the need to screen for CD all family members once a case is diagnosed and shows the possible high rate of congenital CD also in non-endemic areas.

A study on pediatric respiratory tract infections in hospitalised children from Chennai

Globally, acute respiratory tract infections (ARTIs) are the leading cause of childhood morbidity and mortality. However, these infections remain poorly understood due to absence of affordable and effective diagnostic tools. In this study, viral acute respiratory tract infections were studied in hospitalised children up to 5 years of age (n = 256) using a commercial multiplex real time PCR. RSV (45.69%%), RV (17.88%), HBoV (7.95%), Influ B (7.28%), HMPV (6.6%), HPIV3 (5.96%) and Influ A virus (3.97%) were the common etiological agents detected. There was no significant correlation between the clinical signs and symptoms in patients with and without a viral aetiology. Multiplexed real time PCR is an important tool for early detection of viral agents of paediatric ARTI.

Genetic diversity of norovirus associated with outbreaks in school children with acute gastroenteritis in Changzhou, China, 2018-2019.

Norovirus is one of the major causes of outbreaks and sporadic cases of acute gastroenteritis in school children. Obtaining local genotype diversity information regarding norovirus is important for developing and evaluating prevention strategies of the transmission of this virus in school children. Clinical specimens, obtained from the routine acute gastroenteritis surveillance network from 2018 to 2019, were primarily tested using commercial real-time PCR Kit. Samples with Ct value less than 25 were selected and used for complete genome sequencing and those with Ct value between 25 and 30 were selected and used for he partial VP1 and RdRp regions sequencing. Phylogenetic trees of the viral genome were constructed by using the neighbor-joining method with bootstrap analysis of 1000 replicates in MEGA 6.0. Epidemiological surveillance of acute intestinal infections (n = 384) showed high-level detection (73.18%) of human norovirus in school endemic acute gastroenteritis events in Changzhou, with obvious epidemic characteristics in autumn and winter. Through genotyping, it was found that 93.12% of norovirus were GII, including GII.2, GII.3, GII.4, GII.6, GII.7, and GII.17. By October 2019, two norovirus genotypes, GII.4[P31] and GII.17[P17], became the preponderant epidemic strains. Phylogenetic analysis of the new GII.17[P17] complete genomes showed close relationship with Miyagi strain identified in Japan in 2015, and GII.4[P31] showed close relationship with Jinan strain indentified in China in 2017. The study highlights the emerging role of GII.4[P31] and GII.17[P17] in causing endemic acute gastroenteritis outbreaks at school children, in Changzhou, China in 2019.

Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis

TaqMan probe-based commercial real-time (RT) PCR kits are expensive but most frequently used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections needs to meet the challenge of testing more persons at a reasonable cost. This study developed a simple and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay targeting two virus-specific genes along with a host-specific internal control. A total of 180 randomly selected samples portioning into two subsets based on crude and high-quality RNA extraction were used to compare this assay with a nationwide available commercial kit (Sansure Biotech Inc., (Hunan, China)), so that we could analyze the variation and validity of this in-house developed method. Our customized-designed primers can specifically detect the viral RNA likewise Sansure. We separately optimized SYBR Green RT-PCR reaction of N, E, S, and RdRp genes based on singleplex melting curve analysis at the initial stage. After several rounds of optimization on multiplex assays of different primer combinations, the optimized method finally targeted N and E genes of the SARS-CoV-2 virus, together with the β-actin gene of the host as an internal control. Comparing with the Sansure commercial kit, our proposed assay provided up to 97% specificity and 93% sensitivity. The cost of each sample processing ranged between ~2 and ~6 USD depending on the purification level of extracted RNA template. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in low-income countries.

A comparative study of commercial real-time reverse transcription PCR kits for forensic body fluid identification

ABSTRACT Identifying the type(s) of body fluid present in forensic casework exhibits can assist with scene reconstruction and indicate potential activity types. Confirmatory body fluid identification can be achieved using mRNA-based profiling assays. This commonly involves endpoint reverse-transcription PCR (RT-PCR) with capillary electrophoresis (CE) detection. In comparison, real-time quantitative RT-qPCR is more sensitive, quantitative, and does not require post-PCR processing. We have developed real-time RT-qPCR assays for forensic body fluid identification. This study compared the performance (sensitivity, PCR efficiency, precision) of five real-time RT-qPCR kits across circulatory blood, buccal, semen, and vaginal fluid samples with normal and extended (3-step) PCR cycling. An objective scoring system for the experimental performance parameters was considered along with other features of the commercial kits. Statistical analysis by ANOVA and post-hoc Tukey of the slope estimates, which relate to PCR efficiency, revealed that many observed differences were insignificant (p > 0.05). Sensitivity and precision were also similar across most kits and PCR cycling protocols. Using the scoring system, the five highest performing kit and cycling combinations were: TaqMan-extended, followed by the TaqPath-normal, QuantiNova-extended, QuantiNova-extended, then UltraPlex-normal. Based upon high performance, room temperature set-up, and multiplexing capability, the UltraPlex kit was selected for our body fluid identification assays.

Development of a Real-Time Recombinase Polymerase Amplification Assay for the Rapid Detection of African Swine Fever Virus Genotype I and II.

African swine fever (ASF) is a contagious viral disease in pigs and wild boars which poses a major threat to the pig industry. Rapid and accurate diagnosis is necessary to control ASF. Hence, we developed a rapid diagnostic method using a recombinase polymerase amplification (RPA) assay targeting the conserved sequences of CP204L (p30) thatcan rapidly detect ASF virus (ASFV) genotype strains I and II. The lower detection limit of the real-time RPA assay was 5 × 101 copies per reaction. The real-time RPA assay effectively detected ASFV isolates and clinical specimens belonging to ASFV genotypes I and II. The sensitivity and specificity of the assay were 96.8% (95% confidence interval (CI): 83.3–99.9) and 100% (95% CI: 88.4–100.0), respectively. The agreement between the real-time RPA assay and a reference commercial real-time quantitative polymerase chain reaction (qPCR) was 100%. The real-time RPA assay had a detection time of 6.0 min (95% CI: 5.7–6.2), which was significantly shorter than that of qPCR (49 min; 95% CI: 47.4–50.6; p < 0.001). Thus, the developed real-time RPA assay is a rapid and accurate diagnostic tool for detecting ASFV genotypes I and II.

SARS-CoV-2 in the knee joint: a cadaver study.

Despite the considerable research efforts being made to learn more about COVID-19, little is known about the presence of SARS-CoV-2 genetic material in biological fluids other than respiratory droplets, blood, and feces. The aim of this post-mortem study was to assess the presence of SARS-CoV-2 RNA in the knee synovial fluid, synovial tissue, and bone tissue of COVID-19 patients in order to discover whether the joint is a possible route of transmission during orthopaedic surgical procedures, and clarify the possible role of SARS-CoV-2 as a directly arthritogenic virus. Post-mortem synovial fluid, synovial tissue and bone tissue samples were collected from the knees of five patients who died of COVID-19 in our hospital between September and October 2020, and analysed for the presence of SARS-CoV-2 using a commercial real-time polymerase chain reaction (RT-PCR) panel. Quantitative RT-PCR was used to test post-mortem nasopharyngeal swabs of all of the patients. No SARS-CoV-2 RNA was detected in any of the knee samples, despite the positivity of the throat swab. Our findings indicate that SARS-CoV-2 was not detected in knee synovial fluid, synovial membrane or bone. This makes it unlikely that these are potential sources of contagion, and suggests that SARS-CoV-2 is not directly arthritogenic.

BsmI gene polymorphism of vitamin D receptor in obese Egyptian male medical students and its relationship with vitamin D deficiency

BackgroundVitamin D regulates cell proliferation and differentiation and exhibits immunoregulatory, antiangiogenic, and antioxidant characteristics. Vitamin D deficiency and vitamin D receptor (VDR) gene polymorphisms have been associated with an increased risk of metabolic syndrome components such as abdominal obesity, with both vitamin D deficiency and VDR gene polymorphisms contributing to its development. Obesity, metabolic disorders, and bone mass defects are associated with VDR alleles. The importance of VDR in the etiology of obesity has been related to the existence of the VDR polymorphisms FokI, BsmI, ApaI, and TaqI. VDR expression in adipocytes affects energy metabolism and aids in obesity development.ObjectiveThe aim of this study was to examine a possible association between BsmI (B, b allele) polymorphism in the VDR gene and vitamin D status in obese Egyptian male medical students.Patients and methodsThis study was based on a comprehensive experiment known as “Nutritional Assessment of Medical Educational Students (NAMES)” that was completed in 2018 with 700 healthy participants (men and women). In total, out of the 700 participants 30 healthy men were enrolled in this study who matched our inclusion criteria. They attended Ain Shams University Hospital in Cairo between April 2019 and November 2020. This study was conducted at the Clinical Pathology Department of Ain Shams University Hospital, which is a part of the Faculty of Medicine. Participants were divided into two groups: Group I consisted of 12 obese men with nondeficient vitamin D levels (> 20 ng/dl), and Group II consisted of 18 obese men with deficient vitamin D levels (20 ng/dl). Peripheral blood samples were collected into EDTA tubes from all groups, and DNA was extracted and purified using spin purification for PCR (QIA amp DNA Mini Kit). A commercial real-time polymerase chain reaction (PCR) kit with predesigned TaqMan probes was used to analyze the single nucleotide polymorphism (SNP) (BsmI) rs 1544410 in the VDR gene.ResultsNo statistically significant relationship was observed between vitamin D levels and all InBody bioelectrical impedance characteristics, as well as BsmI gene polymorphism and all InBody bioelectrical impedance parameters (P > 0.05). Regarding Bb and BB genotypes, no statistically significant difference was observed between Groups I and II. The prevalence of BB genotype was higher in vitamin-D-deficient individuals, and Bb genotype was more common among obese participants than BB genotype, which showed a higher prevalence of the “b” gene; however, these were not significant. Iron profile (iron level, ferritin level, TIBC, and transferrin saturation) and BsmI gene polymorphism showed no statistically significant relationship (P > 0.05). Glycated hemoglobin (HbA1c) level and BsmI gene polymorphism showed a statistically significant relationship (P = 0.002), with a higher mean value among Bb genotype carriers than among BB genotype carriers. Eosinophil count showed a statistically significant difference between BB and Bb genotype carriers (P = 0.045), with a higher mean value among Bb genotype carriers than among BB genotype carriers. Bsml gene polymorphism showed no statistically significant relationship with any other complete blood count parameters (P > 0.05.)ConclusionThere was no significant relationship between VDR gene polymorphism (BsmI) and body mass index (BMI) nor between the different InBody bioelectrical impedance parameters. We noticed the prevalence of BB genotype among vitamin-D-deficient obese students and the frequency of the “b” allele among obese candidates according to the findings of our study. There was also no significant relationship between BsmI gene polymorphism and vitamin D levels. However, BsmI gene polymorphism and HbA1c levels and eosinophil count showed a relationship, which requires further investigation.

Epidemiological survey and risk factors associated with hepatitis E virus in small ruminants in southern Spain.

Autochthonous cases of hepatitis E (HE) associated with zoonotic genotypes HEV‐3 and HEV‐4 have significantly increased in industrialized countries over the last decade. Suidae are generally recognized as the main reservoirs of these genotypes. Susceptibility to HE virus (HEV) infection and zoonotic potential have also been confirmed in other species, including sheep and goat. However, the information about their role in the epidemiology of HEV remains very scarce. The objective of this study was to assess the prevalence, spatial distribution and risk factors associated with HEV exposure in sheep and goats in southern Spain, the country with the highest census of small domestic ruminants in the European Union. Blood samples from 240 sheep and 240 goats were collected between 2015 and 2017. Sera were analysed in parallel using a commercial double‐antigen ELISA and real‐time PCR. A total of 38 (7.9%; 95%CI: 5.5–10.3) out of 480 sampled animals showed anti‐HEV antibodies. By species, the seroprevalences found in sheep and goats were 2.1% (5/240; 95%CI: 0.3–3.9) and 13.8% (33/240; 95%CI: 9.4–18.1) respectively. Anti‐HEV antibodies were found on 19 (59.4%; 95%CI: 42.4–76.4) of the 32 sampled farms. The GEE model showed that species (goat) and number of small ruminants in the farm (≤348 animals and ≥538 animals) were risk factors potentially associated with HEV exposure in small ruminants in the study area. HEV RNA was not detected in any of the 480 (0.0%; 95%CI: 0.0–0.8) tested animals. Our results confirm that sheep and goats are naturally, but not equally exposed to HEV and indicate the widespread spatial distribution of HEV among small ruminant populations in southern Spain. Further studies are required to elucidate the role of sheep and goat in the epidemiology of HEV and their potential implications for public health.

Comparison of the sensitivity, specificity, correlation and inter-assay agreement of eight diagnostic in vitro assays for the detection of African swine fever virus.

With the recent spread of African swine fever (ASF) in Europe, Asia and the Caribbean region, after being endemic for decades in Africa, PCR-based commercial kits and various master mixes are increasingly being used in addition to the Office International des Epizooties-recommended protocol from King etal. (World Organization for Animal Health). Often, the availability and cost of commercial kits or master mixes can be a limiting factor for diagnostic laboratories, in addition to the requirements for transportation and storage of temperature-sensitive reagents in remote areas. In such cases, alternatives should be ready to maximize surveillance and mining of ASF. To evaluate alternatives, we tested five commercial quantitative real-time PCR (qPCR) master mixes from Applied Biosystems, Bio-Rad, Biotechrabbit, Promega and Qiagen using the same primers and probe mix derived from the King etal.'s protocol for the sensitivity, specificity, correlation and inter-assay agreement. We further included three ad hoc molecular diagnostic kits (VetMax™ African Swine Fever Virus Detection Kit [Applied Biosystems], ID Gene African Swine Fever Duplex [ID-Vet] and Virotype ASF PCR Kit [Qiagen/Indical]). The limit of detection (LOD) was assessed for each assay. The comparative study panel comprised 83 archived DNA samples from ASF virus (ASFV) clinical samples, belonging to five different genotypes from outbreaks in 16 countries in Asia and Africa. The analytical specificity was assessed against a panel of swine pathogens. The LOD ranged from 13 to 41 gene copies per reaction; VetMax ™ African Swine Fever Virus Detection Kit from Applied Biosystems exhibited the lowest detection limit (13 gene copies per reaction) and iQ Supermix from Bio-Rad the highest detection limit (41 gene copies per reaction). Cq values obtained from the lowest dilution, in which all replicates (n=25) could still be amplified (50 gene copies per reaction), were not significantly different between kits using Kruskal-Wallis test. Inter-assay agreement was assessed using statistical test Fleiss-Kappa and was shown to be excellent in all cases. Agreement using statistical test Bland-Altman was good for samples with Cq values<25 and moderate for Cq values>25. We conclude that all the assays evaluated in this study can be used for the routine detection of ASFV.

Developing a multiplex loop-mediated isothermal amplification assay (LAMP) to determine severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus.

Severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus are endemic zoonotic diseases that pose significant public health threats in East Asia. As these two diseases share common clinical features, as well as overlapping disease regions, it is difficult to differentiate between SFTS and scrub typhus. A multiplex reverse-transcription loop‑mediated isothermal amplification (RT-LAMP) assay was developed to detect large segments and GroES genes for SFTS virus (SFTSV) and Orientia tsutsugamushi (OT). The performance of the RT-LAMP assay was compared and evaluated with those of commercial PowerChek™ SFTSV real-time PCR and LiliF™ TSUTSU nested PCR for 23 SFTS and 12 scrub typhus clinical samples, respectively. The multiplex SFTSV/OT/Internal control (IC) RT-LAMP assay showed comparable sensitivity (91.3%) with that of commercial PowerChek™ SFTSV Real-time PCR (95.6%) and higher sensitivity (91.6%) than that of LiliF™ TSUTSU nested PCR (75%). In addition, the multiplex SFTSV/OT RT-LAMP assay showed 100% specificity and no cross-reactivity for blood from uninfected healthy patients and samples from patients infected with other fever viruses. Thus, the multiplex SFTSV/OT/IC RT-LAMP assay could serve as a useful point-of-care molecular diagnostic test for SFTS and scrub typhus.

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176€, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I2S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled high-accuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform high-resolution thermal shift assays (TSA) from exemplary biological applications.