Multiplex Real-time RT-PCR Research Articles

A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses

Avian influenza viruses (AIVs) have caused a large number of epidemics in domestic and wild birds, and even posed a health challenge to humans. Highly pathogenic AIVs have attracted the most public attention. However, low pathogenic AIVs, including H4, H6, and H10 subtype AIVs, have spread covertly in domestic poultry, without obvious clinical signs. The emergence of human infections with H6 and H10 AIVs and the evidence of seropositivity of H4 AIV in poultry-exposed individuals indicated that these AIVs sporadically infect humans and could cause a potential pandemic. Therefore, a rapid and sensitive diagnostic method to simultaneously detect Eurasian lineage H4, H6, and H10 subtype AIVs is urgently required. Four singleplex real-time RT-PCR (RRT-PCR) assays were established based on carefully designed primers and probes of the conserved regions of the matrix, H4, H6, and H10 genes and combined into a multiplex RRT-PCR method to simultaneously detect H4, H6, and H10 AIVs in one reaction. The detection limit of the multiplex RRT-PCR method was 1–10 copies per reaction when detecting standard plasmids, and showed no cross-reaction against other subtype AIVs and other common avian viruses. Additionally, this method was suitable to detect the AIVs in samples from different sources, the results of which showed high consistency with virus isolation and a commercial influenza detection kit. In summary, this rapid, convenient, and practical multiplex RRT-PCR method could be applied in laboratory testing and clinical screening to detect AIVs.

Evaluation of saliva and nasopharyngeal swab sampling for genomic detection of SARS-CoV-2 in children accessing a pediatric emergency department during the second pandemic wave.

SARS-CoV-2 infection is mainly detected by multiplex real-time RT-PCR from upper respiratory specimens, which is considered the gold-standard technique for SARS-CoV-2 infection diagnosis. A nasopharyngeal (NP) swab represents the clinical sample of choice, but NP swabbing can be uncomfortable to the patients, especially for pediatric-age participants, requires trained healthcare personnel, and may generate an aerosol, increasing the intrinsic exposure risk of healthcare workers. The objective of this study was to compare paired NP and saliva samples (SS) collected from pediatric patients to evaluate whether the saliva collection procedure may be considered a valuable alternative to the classical NP swab (NPS) sampling in children. In this study, we describe a SARS-CoV-2 multiplex real-time RT-PCR protocol for SS, comparing the results with the paired NPS specimens from 256 pediatric patients (mean age 4.24 ± 4.40 years) admitted to the hospital emergency room of Azienda Ospedaliera Universitaria Integrata (AOUI), Verona, and randomly enrolled between September 2020 and December 2020. The saliva sampling demonstrated consistent results when compared to NPS use. The SARS-CoV-2 genome was detected in 16 out of 256 (6.25%) NP samples, among which 13 (5.07%) were positive even when paired SS were analyzed. Moreover, SARS-CoV-2-negative NPS and SS were consistent, and the overall concordances between NPS and SS were detected in 253 out of 256 samples (98.83%). Our results suggest that saliva samples may be considered a valuable alternative to NPS for SARS-CoV-2 direct diagnosis with multiplex real-time RT-PCR in pediatric patients.

Мониторинг минимальной остаточной болезни при остром миелоидном лейкозе с перестройками гена KMT2A: сравнение методов мультиплексной и моноплексной ОТ-ПЦР в режиме реального времени

Острый миелоидный лейкоз (ОМЛ) занимает 15–20% в структуре детских острых лейкозов. Несмотря на успехи, достигнутые за последние годы, терапия ОМЛ остается трудной задачей. Важную роль в определении оптимальной тактики терапии играет определение уровня минимальной остаточной болезни (МОБ). Для ОМЛ с перестройками гена KMT2A одним из наиболее чувствительных методов детекции МОБ является количественная ПЦР. Перестройки гена KMT2A представляют собой крайне гетерогенную группу, в связи с чем для их первичной детекции применяют различные молекулярно-биологические методы, включая длинную инвертированную ПЦР, мультиплексную ПЦР и таргетное высокопроизводительное секвенирование, что позволяет не только подтвердить наличие перестройки гена KMT2A, но и уточнить характер этой перестройки. В дальнейшем для мониторинга МОБ может применяться как мультиплексная, так и моноплексная количественная ПЦР. Целью работы являлось сравнение методов мультиплексной и моноплексной ОТ-ПЦР в режиме реального времени в контексте мониторинга МОБ у пациентов с KMT2A-позитивными ОМЛ. В исследование было включено 20 пациентов с KMT2A-позитивным ОМЛ, проходивших диагностику в лаборатории цитогенетики и молекулярной генетики НМИЦ ДГОИ имени Дмитрия Рогачева. Определение экспрессии химерного транскрипта выполнено суммарно в 37 точках, из них 14 – исследование на инициальном материале, 23 – исследование на материале в контрольных точках, предусмотренных протоколом лечения. В 33 случаях химерный транскрипт детектировался обоими методами, в 4 случаях – только при использовании моноплексной ОТ-ПЦР в режиме реального времени. При использовании моноплексной ОТ-ПЦР отношение копийности химерного транскрипта к копийности контрольного гена было достоверно выше, нежели при использовании мультиплексной ОТ-ПЦР (n=33, p<0,001). Проведенное сравнение демонстрирует, что моноплексная ОТ-ПЦР в режиме реального времени является не только более специфичным, но и более чувствительным методом детекции МОБ. Acute myeloid leukemia (AML) occupies 15–20% in the structure of childhood acute leukemia. Despite the advances made in recent years, the treatment of AML remains a challenge. The level of minimal residual disease (MRD) plays an important role in determining of the optimal therapy tactics. For AML with KMT2A gene rearrangements, quantitative PCR is one of the most sensitive methods for MRD detection. KMT2A rearrangements are extremely heterogeneous, and therefore, for their primary detection, various molecular methods are used, including long-distance inverse PCR, multiplex PCR, and targeted high-throughput sequencing, which enables not only to confirm KMT2A rearrangement presence, but also to clarify its structure. Subsequently, both multiplex and monoplex quantitative PCR can be used for MRD monitoring. The purpose of the work was to compare methods of multiplex and monoplex real-time RT-PCR in the context of MRD monitoring in patients with KMT2A-positive AML. The study included 20 patients with KMT2A-positive AML who underwent diagnostics in the laboratory of cytogenetics and molecular genetics in Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The chimeric transcript expression analysis was performed at 37 points, including 14 initial and 23 MRD samples as determined by the treatment protocol. In 33 cases, the chimeric transcript was detected by both methods, and in 4 cases, only by real-time monoplex RT-PCR. When using monoplex RT-PCR, the copy ratio of the chimeric transcript to the copy number of the control gene was significantly higher than when using multiplex RT-PCR (n=33, p<0.001). This comparison demonstrates that real-time monoplex RT-PCR is not only more specific, but also more sensitive for PCR- based MRD detection.

A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan.

The rapid identification of Influenza A virus and its variants, which cause severe respiratory diseases, is imperative to providing timely treatment and improving patient outcomes. Conventionally, two separate assays (total test duration of up to 6 h) are required to initially differentiate Influenza A and B viruses and subsequently distinguish the pdm H1N1 and H3N2 serotypes of Influenza A virus. In this study, we developed a multiplex real-time RT-PCR method for simultaneously detecting Influenza A and B viruses and subtyping Influenza A virus, with a substantially reduced test duration. Clinical specimens from hospitalized patients and outpatients with influenza-like symptoms in Eastern Taiwan were collected between 2011 and 2015, transported to Hualien Tzu Chi Hospital, and analyzed. Conventional RT-PCR was used to subtype the isolated Influenza A viruses. Thereafter, for rapid identification, the multiplex real-time RT-PCR method was developed and applied to identify the conserved regions that aligned with the available primers and probes. Accordingly, a multiplex RT-PCR assay with three groups of primers and probes (MAF and MAR primers and MA probe; InfAF and InfAR primers and InfA probe; and MBF and MBR primers and MB probe) was established to distinguish these viruses in the same reaction. Thus, with this multiplex RT-PCR assay, Influenza B, Influenza A pdm H1N1, and Influenza A H3N2 viruses were accurately detected and differentiated within only 2.5 h. This multiplex RT-PCR assay showed similar analytical sensitivity to the conventional singleplex assay. Further, the phylogenetic analyses of our samples revealed that the characteristics of these viruses were different from those reported previously using samples collected during 2012-2013. In conclusion, we developed a multiplex real-time RT-PCR method for highly efficient and accurate detection and differentiation of Influenza A and B viruses and subtyping Influenza A virus with a substantially reduced test duration for diagnosis.

Multiplex RT-qPCR Application in Early Detection of Bovine Respiratory Disease in Healthy Calves

Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.

Tailored Multiplex Real-Time RT-PCR with Species-Specific Internal Positive Controls for Detecting SARS-CoV-2 in Canine and Feline Clinical Samples

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.

Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 100 copies/μL for the multiplex qRT-PCR and 3.20 × 10-1 copies/μL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.

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.

Development and Validation of ScriptTaq COVID PCR: An In-House Multiplex rRT-PCR for Low-Cost Detection.

The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.

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

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.

Genetic Evolution Analysis and Host Characteristics of Hantavirus in Yunnan Province, China.

For a long time, the epidemic situation of hemorrhagic fever with renal syndrome (HFRS) caused by hantavirus (HV) in Yunnan Province of China has been relatively severe. The molecular epidemiology and host characteristics of HV in Yunnan Province are still not completely clear, and the systematic and long-term investigation of the epidemic area is very limited. In this study, a total of 488 murine-shaped animals were captured in the three regions of Mile City, Mangshi City and Lianghe County in Yunnan Province, and then the type of HV was identified by multiplex real-time RT-PCR and sequenced. The results indicate that 2.46% of the murine-shaped animal specimens were infected with HV. A new subtype of Seoul virus (SEOV) was found in the rare rat species Rattus nitidus in Lianghe County, and the two strains of this new subtype were named YNLH-K40 and YNLH-K53. Through the phylogenetic analysis of this new subtype, it is shown that this new subtype is very similar to the type S5 of SEOV, which is previously described as the main cause for the high incidence of HFRS in Longquan City, Zhejiang Province, China. This new subtype is highly likely to cause human infection and disease. Therefore, in addition to further promoting the improvement of the HV gene database and strengthening the discovery and monitoring of the host animals in Yunnan Province, more attention should be paid to the pathogenic potential of the newly discovered HV type.

Prevalence of viral gastroenteritis pathogens among hospitalized children under 5 year of age in the process of introducing the RV vaccine in the routine immunization

Rotavirus (RV), Norovirus (NV), Sapovirus (SaV) and Astrovirus (Ast) are the leading causes of gastroenteritis and mortality in children worldwide. RV vaccines help to reduce the burden of diarrhea while the importance of other viruses is increasing. Therefore, we conducted a cross-sectional descriptive study to determine prevalence of these viruses in hospitalized children under 5 years old in Nam Dinh province with approximately 80% of RV vaccine coverage. We randomly selected 579 (25%) among diarrheal samples collected in 4 district hospitals from Dec. 2017 to Nov. 2019. An ELISA kit was employed to detect RV and the multiplex real-time RT-PCR with specific primers to detect NV, SaV and Ast. These viruses were found among 64.3% of hospitalized children, of which NV and RV were present in 28.5% and 21.4%, respectively, while SaV and Ast were detected in 3.3% and 2.6%, respectively. Co-infections accounted for 8.5%, mainly among RV and 3 other viruses. There is a significant difference in RV and NV positive rates between vaccinated and unvaccinated groups (p &lt; 0.001 and p = 0.008, respectively). This study contributed to the understanding of the potential impact of rotavirus vaccine on other diarheal pathogens and emphasize the need for further epidemiological studies when the nationwide RV vaccination in Vietnam is implementing.

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.

Evaluation of Feedstuffs as a Potential Carrier of Avian Influenza Virus between Feed Mills and Poultry Farms.

The present study was conducted to assess the potential vector role of feedstuffs for the area spreading of avian influenza virus (AIV). Firstly, feed samples were collected from commercial poultry facilities that experienced highly pathogenic avian influenza (H5N2) in 2014–2015 for AIV testing by a real-time RT–PCR specific for the viral matrix gene. Secondly, feed materials obtained from an AIV-negative farm were spiked with various concentrations of a low pathogenic AIV H5N2. Virus-spiked cell culture media were prepared in the same manner and used for comparison. The spiked feed and media samples were tested by a multiplex real-time RT–PCR ran in a quantitative manner, either immediately or after incubation at −20, 4, 22, and 37 °C for 24, 48, and 72 h. Some of the feedstuffs collected from the poultry facilities or feed mills were positive for AIV RNA but negative by the virus isolation (VI) test, while all the formaldehyde-treated feedstuffs were PCR-negative. In the spiked feeds, the AIV titer was 1–3 logs lower than that in the corresponding media, even when tested immediately after spiking, suggesting that feed might have a negative impact on the virus or PCR detection. The half-life of AIV RNA was shorter at a higher temperature. A significant decay in the viral RNA over time was noted at 37 °C (p < 0.05), suggesting that feedstuffs should be maintained in the cold chain when testing is desired. Furthermore, the thermal degradation of AIV suggests that the heat treatment of feeds could be an alternative to chemical treatment when contamination is suspected. Collectively, the study observations indicate that AIV survivability in feed is relatively low, thus rendering it a low risk.

A Multiplex Crystal Digital PCR for Detection of African Swine Fever Virus, Classical Swine Fever Virus, and Porcine Reproductive and Respiratory Syndrome Virus.

African swine fever (ASF), classical swine fever (CSF), and porcine reproductive and respiratory syndrome (PRRS) are highly infectious diseases of domestic pigs and wild boars. The co-infections of ASF virus (ASFV), CSF virus (CSFV), and PRRS virus (PRRSV) have been reported in different pig farms. Early differential detection and diagnosis of ASFV, CSFV, and PRRSV in the clinical samples is very important for the effective prevention and control of these diseases. A multiplex crystal digital PCR (dPCR) was developed for differential detection of ASFV, CSFV, and PRRSV in this study, targeting p72, 5' untranslated region (UTR), and ORF7 genes, respectively. The different reaction conditions were optimized, and the specificity, sensitivity, and repeatability of the assay were evaluated. The results showed that the multiplex crystal dPCR was able to accurately and differentially detect ASFV, CSFV, and PRRSV with a limit of detection of 4.69 × 10−1 copies/μl, respectively, and could not detect other porcine viruses, i.e., foot-and-mouth disease virus (FMDV), Senecavirus A (SVA), atypical porcine pestivirus (APPV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV). The assay showed excellent repeatability and reproducibility, with coefficients of variation (CV) of the intra- and inter-assay from 0.09 to 1.40%, and from 0.64 to 2.26%, respectively. The 289 clinical samples from different pig herds in Guangxi province, China, were tested by the multiplex crystal dPCR and a reference multiplex real-time quantitative RT-PCR (qRT-PCR) established previously in our laboratory. The positive rates of ASFV, CSFV, and PRRSV were 30.10, 13.49, and 22.49% by the multiplex crystal dPCR, and 24.57, 8.65, and 18.34% by the multiplex qRT-PCR, with coincidence rates of 94.66, 95.16, and 95.84%, respectively. The results indicated that the established multiplex crystal dPCR was a specific, sensitive, and accurate method for the detection and quantification of ASFV, CSFV, and PRRSV. This is the first report on the multiplex dPCR for detecting ASFV, CSFV, and PRRSV.

Analysis of hemagglutinin-neuraminidase gene characteristics of human parainfluenza virus type 3 among children with acute respiratory tract infection in Qingdao city

The purpose was to discuss the infection status of human parainfluenza virus type 3 (HPIV-3) in children with acute respiratory tract infection(ARTI) in Qingdao, Shandong province, and to analyze the gene characteristics of HPIV-3 hemagglutinin-neuraminidase protein (HN). This study was a cross-sectional study. A total of 1 674 throat swab samples were collected randomly from children with ARTI, in the three hospitals (Qingdao Women and Children's Hospital, West Coast Branch of Affiliated Hospital of Qingdao University, Laoshan Branch of Affiliated Hospital of Qingdao University) from January 2018 to December 2019. Multiplex real-time fluorescence RT-PCR was performed to screen HPIV-3 positive specimens. For HPIV-3 positive specimens, nested PCR was used to amplify the full-length HN gene of HPIV-3. The HN gene was sequenced and compared with the representative strains of HPIV-3 in GenBank, and the phylogenetic tree was established. As results, this study collected 1 674 samples, in which there were 90 HPIV-3 positive samples showed and the detection rate was 5.37%. Among positive specimens, the number of samples from children under 6 years old was 88, accounting for 97.78%. HPIV-3 positive cases were mainly distributed in spring and summer. The full-length sequences of 44 HPIV-3 HN genes were obtained by nested PCR method. Sequence alignment and evolutionary analysis showed that the HPIV-3HN gene belonged to the C3a and C3b branches of C3 genotype, with 30 strains of subtype C3a and 14 strains of subtype C3b. The nucleotide and amino acid homology of the amplified 44 strains of the HPIV-3 HN gene in Qingdao were 97.0%-100.0% and 98.5%-100.0%, respectively. In conclusion, from 2018 to 2019, the C3a and C3b branches of HPIV-3 C3 genotype were circulating prevalent in Qingdao, Shandong province. HN gene variation rate was low, but showed certain regional characteristics in evolution.

Multiplex detection of eight different viral enteropathogens in clinical samples, combining RT-PCR technology with melting curve analysis

BackgroundEarly and accurate identification of infection viruses among children can benefit the personalized medical treatment and management, and reduce the future occurrence of serious symptoms. Thus, it is critical to develop a high-throughput multiplex real-time RT-PCR method to improve the accuracy and efficiency in routine clinical lab tests.MethodsWe developed a real time RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus.ResultsStool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction among the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The limit detection was ranged from 1 × 102 to 1 × 105 nucleic acid copies/ml for each diarrhea virus.ConclusionThe RRCMC method is a suitable rapid clinical test for infectious viruses, with the advantages of high-throughput, low cost, high sensitivity and specificity.

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.