Multiplex Real-time RT-PCR Assay Research Articles

Development and validation of highly sensitive multiplex real-time RT-PCR assay for detection of classical swine fever virus genome

Classical swine fever (CSF) remains a challenge for pig farming industrial lover the world despite the measures taken. The last CS case in the Russian Federation was reported in 2020, however, the threat of the disease emerging still persists. A set of anti-epidemic measures including mainly preventive vaccination and annual diagnostic monitoring using molecular-genetic and serological methods is required for CSF virus introduction prevention and rapid eradication of potential disease out breaks. Therefore, areal-time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Therefore, areal time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Modified primers (locked nucleic acids containing conformationally blocked nucleosides) providing a higher affinity to the DNA matrix and physico-chemical stability and a FAM-labeled TaqMan probe were selected for 5’-untranslatedregion of the genome. The following validation parameters were defined: accuracy, repeatability, reproducibility, specificity and sensitivity. For comparative analysis of the developed as say sensitivity, swabs, samples of organs and tissues collected from pigs experimentally infected with an epizootic strain of the classical swine fever virus (spleen, kidney, liver, blood, lymph nodes, rectal and oral smears), animal-contaminated feed and virus-containing material with known virus titres were also tested in parallel with coded test systems No. x1 andx2. The developed assay was shown to have 100% diagnostic sensitivity and detection limit of 0,23 lgCCID50/cm3. Therewith, there sults of analysis of test systems No. x1, x2 based on above parameters were lower that could give rise to false positive real-time RT-PCR results and incorrect diagnosis. Thus, described assay can be used for extensive monitoring of classical swine fever in the Russian Federation

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses

H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1–10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.

Testing the limits of multiplex respiratory virus assays for SARS-CoV-2 at high cycle threshold values: Comparative performance of cobas 6800/8800 SARS-CoV-2 & Influenza A/B, Xpert Xpress SARS-CoV-2/Flu/RSV, and cobas Liat SARS-CoV-2 & Influenza A/B.

Multiplex real-time RT-PCR assays for respiratory pathogens are valuable tools to optimize laboratory workflow and turnaround time. At a time when resurgence of influenza and respiratory syncytial virus (RSV) cases have been widely observed along with continued transmission of SARS-CoV-2, timely identification of all circulating respiratory viruses is crucial. This study evaluates the detection of low viral loads of SARS-CoV-2 by four multiplex molecular assays: Roche cobas 6800/8800 SARS-CoV-2 & Influenza A/B Test, Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV, cobas Liat SARS-CoV-2 & Influenza A/B, and a laboratory-developed test (LDT). Retrospective upper respiratory tract specimens positive for various respiratory viruses at a range of cycle threshold (Ct) values (18-40) were tested by four multiplex assays. Positive and negative percent agreement (PPA and NPA) with validated RT-PCR assays were calculated. A total of 82 samples were assessed, with discordant results observed in a portion of the samples (10/82, 12.2%) where Ct values were >33. The majority of the discordant results (6/10, 60%) were false negatives. Overall, PPA was 100% (58/58) for cobas 6800, 97.4% (38/39) for GeneXpert, 100% (17/17) for Liat, and 90.5% (57/63) for the LDT. PPA for the LDT increased to 92.1% after manual review of amplification curves. Commercial multiplex respiratory virus assays have good performance for samples with medium to high viral loads (Ct values <33). Laboratories should consider appropriate test result review and confirmation protocols to optimize sensitivity, and may consider reporting samples with additional interpretive comments when low viral loads are detected.

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.

External quality assessment for laboratories in pan-India ILI/SARI surveillance for simultaneous detection of influenza virus and SARS-CoV-2.

The Indian Council of Medical Research has set up a nationwide network of 28 laboratories for simultaneous surveillance of influenza virus and SARS-CoV-2 in ILI/SARI patients, using an in-house developed and validated multiplex real-time RTPCR assay. The aim of this study was to ensure the quality of testing by these laboratories by implementing an external quality assessment program (EQAP). For this EQAP, a proficiency test (PT) panel comprising tissue-culture or egg-grown influenza virus and SARS-CoV-2 was developed. The PT panel was distributed to all the participant laboratories, which tested the panel and submitted the qualitative results online to the EQAP provider. The performance of the laboratories was evaluated on qualitative criteria but cycle threshold (Ct) values were also gathered for each sample. On a qualitative basis, all the laboratories achieved the criteria of 90% concordance with the results of the PT panel provider. Ct values of different samples across the laboratories were within ≤ ±3 cycles of the corresponding mean values of the respective sample. The results of this EQAP affirmed the quality and reliability of testing being done for simultaneous surveillance of influenza virus and SARS-CoV-2 in India.

Prevalence and Characterization of Gastroenteritis Viruses among Hospitalized Children during a Pilot Rotavirus Vaccine Introduction in Vietnam.

Rotavirus (RV), norovirus (NoV), sapovirus (SaV), and human astrovirus (HAstV) are the most common viral causes of gastroenteritis in children worldwide. From 2016 to 2021, we conducted a cross-sectional descriptive study to determine the prevalence of these viruses in hospitalized children under five years old in Nam Dinh and Thua Thien Hue provinces in Vietnam during the pilot introduction of the RV vaccine, Rotavin-M1 (POLYVAC, Hanoi, Vietnam). We randomly selected 2317/6718 (34%) acute diarrheal samples from children <5 years of age enrolled at seven sentinel hospitals from December 2016 to May 2021; this period included one year surveillance pre-vaccination from December 2016 to November 2017. An ELISA kit (Premier Rotaclone®, Meridian Bioscience, Inc., Cincinnati, OH, USA) was used to detect RV, and two multiplex real-time RT-PCR assays were used for the detection of NoV, SaV and HAstV. The prevalence of RV (single infection) was reduced from 41.6% to 22.7% (p < 0.0001) between pre- and post-vaccination periods, while the single NoV infection prevalence more than doubled from 8.8% to 21.8% (p < 0.0001). The SaV and HAstV prevalences slightly increased from 1.9% to 3.4% (p = 0.03) and 2.1% to 3.3% (p = 0.09), respectively, during the same period. Viral co-infections decreased from 7.2% to 6.0% (p = 0.24), mainly due to a reduction in RV infection. Among the genotypeable samples, NoV GII.4, SaV GI.1, and HAstV-1 were the dominant types, representing 57.3%, 32.1%, and 55.0% among the individual viral groups, respectively. As the prevalence of RV decreases following the national RV vaccine introduction in Vietnam, other viral pathogens account for a larger proportion of the remaining diarrhea burden and require continuing close monitoring.

Norovirus gastroenteritis in children under-five years hospitalized for diarrhea in two cities of northeast India: A retrospective study

PurposeNorovirus gastroenteritis, known to cause ‘winter vomiting disease’ is increasingly being identified as a major cause of viral gastroenteritis worldwide. The impact and prevalence of this viral disease are lacking in many parts of India including northeast India. This study aimed to determine the prevalence and association of norovirus gastroenteritis among under-five-year-old hospitalized children in two cities in northeast India (Dibrugarh in Assam & Dimapur in Nagaland). Materials and methodsA retrospective analysis of 407 randomly selected diarrheal stool samples was conducted using a commercial multiplex probed-based real-time RT-PCR assay capable of detecting six-viral gastroenteritis pathogens including Norovirus GI, Norovirus GII, Rotavirus, Human Adenovirus, Human Astrovirus, and Sapovirus. ResultsResults showed that norovirus was detected in 18.4% of the samples (75/407; 95% CI: 14.8%–22.5%), with norovirus genogroup II being the predominant group in 97.3% of norovirus cases. A significant association of norovirus diarrhea was found with seasonality, with higher prevalence in colder months compared to warmer months (22.4% vs 9.1%, p-value:0.002). Additionally, 66.7% (50/75) of cases of norovirus gastroenteritis had reported vomiting as the major symptom and had a shorter duration of diarrhea (p-value 0.03). Co-infections with other viral pathogens were seen in 45.9% (187/407) of the cases. The detection of rotavirus was 67.1% (273/407), human adenovirus (45.9%; 187/407), sapovirus and astrovirus (5.9%, 24/407 each), and norovirus GI (0.5%, 2/407) among the cases. ConclusionThis study reports the prevalence of norovirus gastroenteritis in northeast India and further highlights that norovirus gastroenteritis is responsible for substantial cases of hospitalization of under-five years children in the region.

A multiplex real-time RT-PCR assay for the detection of H1, H2 and H3 subtype avian influenza viruses.

Avian influenza viruses (AIVs) are influenza A viruses, of which subtypes H1, H2 and H3 are highly transmissible in poultry and have the risk of transmission to human as well. It is important to establish an accurate, sensitive and convenient means of virus detection. In this study, we developed a multiplex real-time RT-PCR assay based on conserved sequences of the virus hemagglutinin and matrix, and designed primers and probes for the simultaneous and rapid detection of AIV subtypes H1, H2 and H3. We used different subtypes of AIVs and other avian respiratory viruses for evaluation of the specificity of this method. The results showed good sensitivity, specificity and reproducibility. The detection limit was 10-100 copies per reaction. The method also achieved good concordance with the virus isolation method when compared to 81 poultry samples evaluated. It provides a new method for detecting mixed infections of AIVs.

Arbovirus persistence in North-Western Europe: Are mosquitoes the only overwintering pathway?

In some areas in temperate Europe, genomic analyses of mosquito-borne virus outbreaks have revealed the presence of similar virus strains over several years, indicating local overwintering of these viruses. However, it remains unclear how mosquito-borne viruses can persist in winter, when conditions are generally unfavourable for virus circulation. One of the presumed routes of virus persistence is via diapausing mosquitoes. Here, we set out to study whether arbovirus persistence of West Nile virus (WNV), Usutu virus (USUV) and Sindbis virus (SINV) occurs in diapausing mosquitoes in the Netherlands. To this end, mosquito collections were carried out in the winter of 2020 and 2021, in hibernacula located in two areas with previously observed WNV and/or USUV activity. In total, we collected 4200 mosquitoes belonging to four species (Culex pipiens, Culiseta annulata, Anopheles maculipennis s.l., and Culex territans), which were pooled in 490 monospecific pools. These pools were subjected to WNV-, USUV- and SINV-screening using a multiplex real-time RT-PCR assay. All mosquito pools tested negative for the presence of WNV, USUV and SINV RNA. Consequently, we did not find evidence of arbovirus persistence in diapausing mosquitoes in the Netherlands, even though USUV and WNV have re-appeared in birds and/or mosquitoes during the summer seasons of 2020–2022. Concluding, given the persistence of USUV and WNV in the Netherlands and SINV in other temperate regions, this study highlights the importance of further research on (alternative) arbovirus overwintering routes.

Multiplex SYBR Green real-time RT-PCR assays for the improved detection of viral pathogens in horses: equine infectious anemia, West Nile and Japanese encephalitis viruses

Multiplex SYBR Green real-time RT-PCR assays for the improved detection of viral pathogens in horses: equine infectious anemia, West Nile and Japanese encephalitis viruses

Molecular Survey on A, B, C and New Avian Metapneumovirus (aMPV) Subtypes in Wild Birds of Northern-Central Italy.

Simple SummaryAvian metapneumovirus (aMPV) is a common pathogen in poultry and has been detected in wild birds, suggesting the possible role in viral dissemination. A feature of aMPV is its genetic and antigenic variability, which has allowed the identification of various subtypes of the virus with different characteristics in terms of host tropism. Two new subtypes of aMPV were recently identified in gulls and parakeets. We aimed to explore the epidemiology of old and new aMPV subtypes in wild birds. Samples were collected in Italy during the surveillance of avian influenza in wild species and were tested with two multiplex real time RT-PCRs that were able to detect and distinguish the aMPV subtypes (A, B, C, gull, and parakeet subtypes). All of the individuals were negative, except for one mallard that was positive for aMPV subtype C. The M and G genes of this strain were molecularly characterized and revealed similarities with Chinese and European strains, including an Italian sequence that was previously detected in a widgeon. These findings confirm the susceptibility of mallards, which are closely related to domestic species, highlighting the importance of the epidemiological monitoring of aMPV circulation.Recent insights into the genetic and antigenic variability of avian metapneumovirus (aMPV), including the discovery of two new subtypes, have renewed interest in this virus. aMPV causes a well-known respiratory disease in poultry. Domestic species show different susceptibility to aMPV subtypes, whereas sporadic detections in wild birds have revealed links between epidemiology and migration routes. To explore the epidemiology of aMPV in wild species, a molecular survey was conducted on samples that were collected from wild birds during avian influenza surveillance activity in Italy. The samples were screened in pools by multiplex real time RT-PCR assays in order to detect and differentiate subtypes A, B, C, and those that have been newly identified. All the birds were negative, except for a mallard (Anas platyrhynchos) that was positive for aMPV subtype C (sampled in Padua, in the Veneto region, in 2018). The sequencing of partial M and full G genes placed the strain in an intermediate position between European and Chinese clusters. The absence of subtypes A and B supports the negligible role of wild birds, whereas subtype C detection follows previous serological and molecular identifications in Italy. Subtype C circulation in domestic and wild populations emphasizes the importance of molecular test development and adoption to allow the prompt detection of this likely emerging subtype.

SARS-CoV-2 genome RNA detection based on multiplex Real-Time RT-PCR assay

Objectives : This work aimed to develop a multiplex Real-Time RT-PCR assay to detect the SARS-CoV-2 virus in a biological sample. Methods : Multiplex Real-Time one-step RT-PCR was performed compared with the singleplex CDC protocol. For the detection of the N1 and N2 viral regions and host human RNase P gene, the fluorescent dyes VIC-BHQ-1, FAM-BHQ-1, and TAMRA-BHQ-2, respectively, were tested in a one-tube triplex reaction. The swabs from 334 nasopharyngeal samples were tested in comparisons with the singleplex method, and a pUC18 plasmid was constructed with concatenating regions from nucleocapsid and RNAse P gene as a positive control. Results : Paired data between the two assays presented a positive correlation. Comparative data demonstrated that the qRT-PCR multiplex was an efficient method presenting 83% of concordance with the singleplex, and the RNA from SARS-CoV-2 was detected in 31 and 41%, respectively. In addition, in 3% of the samples, the viral RNA was only detected in the multiplex. Conclusions : A fast, accurate, and low-cost method for detecting SARS-CoV-2 was obtained, highlighting the choice of conventional fluorophores and probes assisting in epidemiological surveillance and the clinical management of this serious public health disease.

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

BackgroundThe COVID‐19 pandemic caused by SARS‐CoV‐2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real‐time RT‐PCR are critical for the detection of SARS‐CoV‐2 in clinical specimens from patients suspected of COVID‐19.ObjectiveWe aimed to establish and validate an in‐house real‐time RT‐PCR for the detection of SARS‐CoV‐2.MethodologyPrimers and probes sets in our in‐house real‐time RT‐PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real‐time RT‐PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance.ResultsThe limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of C t values observed between two tests with r 2 = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively.ConclusionIn the present study, we established and validated an in‐house real‐time RT‐PCR for molecular detection of SARS‐CoV‐2 in a resource‐limited country.

Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of rubella virus

Rubella surveillance in elimination setting relies on rapid molecular detection of the virus. In this study a multiplex real-time RT-PCR assay for the detection of rubella virus was validated. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the rubella viral coding regions for envelope glycoprotein E1 and non-structural p150 protein, and an endogenous control (human RNaseP). Using dilution series of synthetic RNAs, the limits of detection were determined to be at least 50 copies of rubella RNA. The assay is reproducible with low intra-assay and inter-assay coefficients of variation for both the E1 and the p150 targets. After testing 62 confirmed rubella positive and 165 rubella negative archival clinical samples, the sensitivity and specificity of the multiplex assay were 98.4 and 100 %, respectively. No cross reactivity was identified with clinical specimens positive for eleven other viruses. This multiplex assay successfully detected nine viral genotypes including the predominant genotypes 1E, 1G, 1J, and 2B as well as the 1a vaccine genotype.

Presence of SARS-CoV-2 in the CSF of Guillain-Barré Syndrome Patients Requires Validation

To the Editors: We appreciate the interest in our article1 and the opportunity to respond to the comments. In the published report, we present a pediatric case of COVID-19-associated GBS with detection of SARS-CoV-2 in the cerebrospinal fluid (CSF). In this case, the Quick-DNA/RNA Viral MagBead (Zymo Research Corp, Irvine, CA) extraction kit on the automated KingFisher Flex Purification System (Thermo Fisher Scientific, Waltham, MA) was used for nucleic acid extraction and the Allplex nCoV-2019 kit (Seegene, Inc., Seoul, South Korea) for gene amplification. Moreover, appropriate positive, negative and internal controls were used to add confidence in the results. The multiplex real-time RT-PCR assay used in this case has a limit of detection of 4167 copies/ml and a sensitivity of 100 copies/reaction. Target genes amplified within ≤40 cycle threshold were considered detected, and the patient had a positive result for the presence of SARS-CoV-2 RNA in the CSF. Regarding the possibility of repeating the CSF virus investigation during follow-up, we decided not to perform invasive procedures with exclusively academic purpose, as this would not change the therapeutic approach. In addition, brain magnetic resonance imaging (MRI) was normal and the patient did not show any signs of brain involvement suggestive of Bickerstaff encephalitis, such as external ophthalmoplegia or disturbance of consciousness. Respiratory muscles were not involved nor did the patient had autonomic dysfunction. The symptoms presented by the patients were explained in the article. Finally, it is worth remembering that during the recent Zika and Chikungunya epidemics, viral RNA was also found in CSF of patients with Guillain-Barré syndrome, as well as the presence of IgM and IgG.2 According to Parra et al,3 arbovirus-related GBS may be caused by direct infection or parainfectious nerve damage, due to the short time between onset of infectious and neurologic symptoms. Although direct viral invasion is a less likely pathophysiologic mechanism for a disease classically defined as immune-mediated, the presence of SARS-CoV-2 RNA in CSF makes it impossible for us to rule out this hypothesis.

Clinical evaluation of a multiplex real-timeRT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen’s kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05148-1.

A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2.

The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.

Development of singleplex and multiplex real-time (Taqman®) RT-PCR assays for the detection of viruses associated with fig mosaic disease

Singleplex and multiplex real-time (TaqMan®) RT-PCR assays were developed to detect seven fig-infecting viruses, i.e., fig leaf mottle-associated virus 1 (FLMaV-1), fig leaf mottle-associated virus 2 (FLMaV-2), fig mild mottle-associated virus (FMMaV), fig mosaic virus (FMV), fig latent virus 1 (FLV-1), fig cryptic virus 1 (FCV-1) and fig fleck-associated virus (FFkaV). The sensitivity of the newly developed TaqMan® assays was compared with the corresponding conventional RT-PCR (RT-PCR) using 10° to 10−6 serial dilutions of both cDNA and crude fig extracts. The results showed that the Taqman® RT-PCR assays were generally 102 to 103-fold more sensitive than the RT-PCR assays, except in the case of FLV-1 detection, where the two techniques had the same sensitivity. In the multiplex Taqman® RT-PCR, only a maximum of five viruses could be detected simultaneously in naturally infected fig trees, regardless of which combination of the virus-specific probes and primers were used. Both the RT-PCR and Taqman® RT-PCR assays were used in a large-scale survey of 100 field-grown fig trees in Egypt. The results showed the presence of all seven viruses under study, mostly occurring as mixed infections (63 %). The prevalence of infections observed in the tested samples were as follows: FMV (62 %), FFkaV (59 %), FLMaV-2 (32 %), FLV-1 (16 %), FLMaV-1 (14 %), FCV-1 (7%) and FMMaV (4%). FMV was invariably associated with diseased trees that presented mosaic-like symptoms. In the few cases where the mosaic-affected trees were found to be free of FMV, they were found to be infected with a mixture of two or more other viruses.

Enhancer-derived long non-coding RNAs CCAT1 and CCAT2 at rs6983267 has limited predictability for early stage colorectal carcinoma metastasis

Up-regulation of long non-coding RNAs (lncRNAs), colon-cancer associated transcript (CCAT) 1 and 2, was associated with worse prognosis in colorectal cancer (CRC). Nevertheless, their role in predicting metastasis in early-stage CRC is unclear. We measured the expression of CCAT1, CCAT2 and their oncotarget, c-Myc, in 150 matched mucosa-tumour samples of early-stage microsatellite-stable Chinese CRC patients with definitive metastasis status by multiplex real-time RT-PCR assay. Expression of CCAT1, CCAT2 and c-Myc were significantly up-regulated in the tumours compared to matched mucosa (p < 0.0001). The expression of c-Myc in the tumours was significantly correlated to time to metastasis [hazard ratio = 1.47 (1.10–1.97)] and the risk genotype (GG) of rs6983267, located within CCAT2. Expression of c-Myc and CCAT2 in the tumour were also significantly up-regulated in metastasis-positive compared to metastasis-negative patients (p = 0.009 and p = 0.04 respectively). Nevertheless, integrating the expression of CCAT1 and CCAT2 by the Random Forest classifier did not improve the predictive values of ColoMet19, the mRNA-based predictor for metastasis previously developed on the same series of tumours. The role of these two lncRNAs is probably mitigated via their oncotarget, c-Myc, which was not ranked high enough previously to be included in ColoMet19.