A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

Burden of human metapneumovirus infections among children with acute respiratory tract infections attending a Tertiary Care Hospital, Kathmandu.

BACKGROUND: Acute respiratory infections (ARIs) are one of the most common causes of mortality and morbidity worldwide. Every year millions of children suffer from viral respiratory tract infections (RTIs) ranging from mild to severe illnesses. Human Metapneumovirus (HMPV) is among the most frequent viruses responsible for RTIs. However, HMPV infections and their severity among children have not been explored yet in Nepal. PURPOSE: Therefore, the study was focused on HMPV infections and other potential viral etiologies or co-infections using multiplex PCR among children attending Kanti Children's Hospital and assessed the clinical characteristics of the infections as well as found the co-infections. A hospital-based cross-sectional study was designed and a convenience sampling method was used to enroll children of less than 15 years with flu-like symptoms from both outpatients and inpatients departments over three months of the study period. RESULTS: HMPV infection (13.3%) was the most predominant infection among the different viral infections in children with ARIs in Kanti Children's Hospital. The HMPV was more prevalent in the age group less than three years (21.8%). Cough and fever were the most common clinical features present in all children infected with HMPV followed by rhinorrhea, sore throat, and wheezing. HMPV-positive children were diagnosed with pneumonia (42.9%), bronchiolitis (28.5%), upper respiratory tract infections (14.3%), and asthma (14.3%). The prevalence of HMPV was high in late winter (14.3%) followed by early spring (13.5%). CONCLUSIONS: This study provides the baseline information on HMPV and associated co-infection with other respiratory viruses for the differential diagnosis based on molecular methods and also the comparison of clinical presentations among the different respiratory syndromes.

Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus.

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

Neurogenic pulmonary edema following febrile status epilepticus in a 22-month-old infant with multiple respiratory virus co-detection: a case report.

BACKGROUND: Neurogenic pulmonary edema is a rare but serious complication of febrile status epilepticus in children. Comprehensive screening for viral pathogens is seldomly performed in the work-up of febrile children. CASE PRESENTATION: A 22-month-old girl presented with her first episode of febrile status epilepticus, after which she developed acute pulmonary edema and respiratory failure. After the termination of seizure activity, the patient was intubated and managed on mechanical ventilation in the emergency room. The resolution of respiratory failure, as well as the neurological recovery, was achieved 9 h after admission, and the patient was discharged 6 days after admission without any complications. Molecular biological diagnostic methods identified the presence of human coronavirus HKU1, influenza C virus, and human parainfluenza virus 2 from the patient's nasopharyngeal specimens. CONCLUSIONS: Neurogenic pulmonary edema following febrile status epilepticus was suspected to be the etiology of our patient's acute pulmonary edema and respiratory failure. Timely seizure termination and rapid airway and respiratory intervention resulted in favorable outcomes of the patient. Molecular biological diagnostic methods identified three respiratory viruses; however, their relevance and association with clinical symptoms remain speculative.

Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of Zika virus and Chikungunya virus.

Zika virus (ZIKV) and chikungunya virus (CHIKV) are important human pathogens and mosquito-borne arboviruses, which have resembling history, common vectors, circulating regions, and indistinguishable clinical symptoms. Wide geographical range that is suitable for ZIKV and CHIKV transmission underlines the concern about the impact of epidemic and endemic infections on burden of public health. In the present study, a highly sensitive and specific one-step multiplex real-time RT-PCR assay was developed and evaluated for simultaneous detection and quantification of ZIKV and CHIKV. The single reaction assay employs two pairs of primers and two TaqMan probes that differentiate ZIKV and CHIKV infections. The entire viral genomic RNA in vitro transcribed from full-length infectious clones were used to generate the standard curves for absolute quantification in subsequent tests. The detection limit of the one-step multiplex assay was 1 and 0.5 PFU for infectious ZIKV and CHIKV, respectively. The assessment of specificity indicated this assay is highly specific to targeted viruses showing no amplification of a variety of other flaviviruses. Our assay was able to detect geographically separated and phylogenetically diverse strains of ZIKV and CHIKV. On the applicability of monitoring viral multiplication in cells and testing clinical samples, the one-step multiplex assay provided efficient and accurate determination. The one-step multiplex real-time RT-PCR assay offers a valuable tool for detection of ZIKV and CHIKV and potentially contributes to general surveillance and clinical treatment.

Simultaneous detection of chikungunya virus, dengue virus and human pathogenic Leptospira genomes using a multiplex TaqMan® assay.

BACKGROUND: In 2005-2006 a major epidemics of Chikungunya disease occurred in South-West Indian Ocean islands. In Reunion Island, the magnitude of Chikungunya infection related symptoms was high and with over 38% of serological prevalence in the population. This epidemics illustrated the potential threat of emerging arboviral diseases for inhabitants of Reunion Island and elsewhere since vectors are worldwide distributed. A sentinel surveillance network was set-up to detect emerging pathogens associated with fever over 38 °C and in the absence of known etiologic causes. Leptospirosis is caused by a pathogenic spirochete of the Leptospira genus and is an endemic and recurrent seasonal disease of great concern in Reunion Island. To accurately diagnose potentially infected patients and to advise Health authorities on the presence of emerging pathogens, a rapid diagnostic test was needed that could differentiate between these 3 pathogens. METHODS: A one-step multiplex real-time PCR assay was developed that can simultaneously detect RNA of Chikungunya and Dengue viruses and leptospiral DNA with good performance for a routine diagnostic use. RESULTS: Simplex protocols already published were used with key modifications to implement a triplex assay which was set-up with a small reaction volume to improve cost efficiency. CONCLUSIONS: This approach has enabled greater diagnostic capacity in our laboratory. We established a multiplex approach validated and valuable for cost savings, and with the concurrent detection of 3 pathogens of public health concern.

Evaluation of custom multiplex real - time RT - PCR in comparison to fast - track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses.

BACKGROUND: Severe acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD). METHODS: Nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples. RESULTS: Custom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 - 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more. CONCLUSION: The custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses.

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/µL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam.

OBJECTIVE: This study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation. RESULTS: The rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

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.

Positivity of dengue, chikungunya, and Zika infections in women in Northeast Brazil post-Zika epidemic.

The state of Ceará, in the Northeast Region of Brazil, presents the simultaneous circulation of Zika (ZIKV), dengue (DENV) and chikungunya (CHIKV) viruses. In 2017 there were a high number of cases of these three arboviruses, especially CHIKV. Here, we detected the presence of arboviruses ZIKV, DENV and CHIKV and their coinfections in women in endemic regions of the city of Fortaleza, Ceará in a post-Zika epidemic year. Sociodemographic and environmental characteristics associated with arbovirus positivity were also analyzed. Women (n = 1289) between 15 and 39 years old were included. RT-qPCR was performed for virus detection and IgM antibody positivity was also analyzed. One hundred and six (8.3%) participants were positive for one or more arboviruses. Monoinfections (76; 5.9%) were distributed between 22 (1.7%) for ZIKV, 39 (3.1%) for DENV and 15 (1.2%) for CHIKV. Co-infections were detected in 30 (2.3%) of the positive participants and one case with triple infection was found. IgM positivity was found in 2.4% of ZIKV RT-qPCR, 9.6% of DENV and 16.3% of CHIKV. RT-qPCR positivity for arboviruses was associated with low socioeconomic class and presence of a water box sealing in the household. A higher positivity to the three viruses occurred in the month with the lowest wind velocity, which was also preceded by the highest peak of rain and humidity. We identified the simultaneous circulation and co-infection of ZIKV, DENV and CHIKV in Fortaleza in a post-Zika epidemic year. We also highlight the need for continuous epidemiological surveillance combined with molecular diagnostic tools.

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.

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.

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.

A multiplex real-time RT-PCR method for detecting H5, H7 and H9 subtype avian influenza viruses in field and clinical samples.

In recent years, H5 and H7 subtypes of highly pathogenic avian influenza viruses (HPAIVs) have been identified in poultry worldwide, resulting in large economic losses to poultry production. Furthermore, H9N2 low pathogenic AIVs are reported to provide internal genes for generating novel reassortant AIVs, leading to potential pandemic risks. To establish an accurate, sensitive and convenient diagnostic method for H5, H7 and H9 subtype AIVs in Eurasian lineage, four groups of specific primers and probes were designed based on the conserved fragments of M, H5, H7 and H9 genes, and a multiplex real-time RT-PCR (RRT-PCR) method was established. High sensitivity was achieved for the multiplex RRT-PCR approach, with a detection limit of 1-10 copies (plasmid DNA) per reaction. The specificity of the method was evaluated using diverse subtypes of AIVs and other avian respiratory viruses isolated in eastern China over the last 9 years. Compared with virus isolation, a higher consistency was achieved when assessing 135 field samples and 126 clinical samples. The results showed that the multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis.

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 single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease.

Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks.

Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay.

BACKGROUND: In the recent past, arboviruses such as Chikungunya (CHIKV) and Zika (ZIKV) have increased their area of endemicity and presented as an emerging global public health threat. OBJECTIVES: To design an assay for the simultaneous detection of ZIKV, CHIKV and Dengue (DENV) 1-4 from patients with symptoms of arboviral infection. This would be advantageous because of the similar clinical presentation typically encountered with these viruses and their co-circulation in endemic areas. STUDY DESIGN: In this study we have developed and validated a triplex real time reverse transcription PCR assay using hydrolysis probes targeting the non-structural 5 (NS5) region of ZIKV, non-structural protein 4 (nsP4) from CHIKV and 3' untranslated region (3'UTR) of DENV 1-4. RESULTS AND CONCLUSIONS: The 95% LOD by the triplex assay was 15 copies/reaction for DENV-1 and less than 10 copies/reaction for all other viruses. The triplex assay was 100% specific and did not amplify any of the other viruses tested. The assay was reproducible and adaptable to testing different specimen types including serum, plasma, urine, placental tissue, brain tissue and amniotic fluid. This assay can be easily implemented for diagnostic testing of patient samples, even in a high throughput laboratory.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.

Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection.

BACKGROUND: Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection. OBJECTIVES: In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR). STUDY DESIGN: From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua. RESULTS: The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/µL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/µL. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples). CONCLUSIONS: The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.