Multiplex Real-time Reverse Transcriptase-polymerase Chain Research Articles

Prevalence and molecular characterization of rotavirus strains circulating among children with gastroenteritis in Egypt

Background and objectives Human rotavirus (RV) is the main cause of diarrheal disease among children younger than 5 years old, worldwide. The aim of the current study was to investigate the prevalence of RV infections and the genotype distribution of RV in Egypt. Materials and methods A total of 642 fecal samples were collected from children younger than 5 years of age, suffering from acute diarrhea and attending ten regional public hospitals (n=585) and five private clinics (n=57) located in 6 Egyptian cities between February and June 2021. All samples were screened by immunochromatographic assay to determine RV prevalence. Then RV-positive samples were further subjected for detection of G (VP7) and P (VP4) genotypes by seminested multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results Out of the 642 children, RV was detected in 268 (41.7%). Inpatients were more likely to be RV-positive (43.2%) than outpatients (26.3%) and most of the positive samples 215/268 (80.2%) were found in children less than 1 year of age. RV infections were more common in males than females (65.3% vs. 34.7%). The VP7 predominant G type was G3 (31.3%), followed by G8 (20.5%), G1 (7%), mixed G infections (6.3%), G2 (1.9%), G9 (1.9%), G4 (1.5%), and G10 (0.4%). The VP4 predominant P type was P[8] (53.7%), followed by P[4] (16%), P[6] (9.3%), P[9] (6.3%), P[11] (4.5%), P[10] (2.6%), and mixed P infections (1.9%). The dominant VP7/VP4 combination was G3P[8] (24.2%), G8P[8] (10%), G8P[4] (5.2%), G1P[8] (4.5%), G8P[6] (3.3%), and G3P[4] (2.2%). Several other combinations were also identified with detection rates less than 2% of positive RV samples. Mixed genotype combinations and partially typed strains were detected in 31.7% and 7.5%, respectively. Conclusion This study highlights the necessity for continuous epidemiology and surveillance of RVA infection to improve our control and management of RVA infection. Furthermore, due to the lack of a national anti-RV vaccination program, RV remains the main causative agent for acute gastroenteritis in Egyptian children. Therefore, it is important to introduce RV vaccine into the national immunization program in Egypt free of charge to all infants to reduce the burden of RV gastroenteritis.

Enhanced throughput of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR panel by assay multiplexing and specimen pooling

A multiplex real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed based on the same primer and probe sequences of an existing U.S. CDC Emergency Use authorized test panel, targeting SARS-CoV-2 N1, N2 and human RNase P genes in singleplex. Both singleplex and multiplex assays demonstrated linear dynamic ranges of 8 orders of magnitude and analytical limits of detection of 5 RNA transcript copies/reaction. Both assays showed 100 % agreement with 364 previously characterized clinical specimens (146 positive and 218 negative) for detection of SARS-CoV-2 RNA. To further increase testing throughput, 40 positive and 20 negative four-specimen pools were tested by the multiplex assay and showed 97.75 % and 100 % congruence with individual specimen tests, respectively. rRT-PCR assay multiplexing and sample pooling, individually or in combination, can substantially increase throughput of SARS-CoV-2 testing.

Zika virus detection in amniotic fluid and Zika-associated birth defects

Zika virus infection during pregnancy can cause serious birth defects, which include brain and eye abnormalities. The clinical importance of detection of Zika virus RNA in amniotic fluid is unknown. The purpose of this study was to describe patterns of Zika virus RNA testing of amniotic fluid relative to other clinical specimens and to examine the association between Zika virus detection in amniotic fluid and Zika-associated birth defects. Our null hypothesis was that Zika virus detection in amniotic fluid was not associated with Zika-associated birth defects. We conducted a retrospective cohort analysis of women with amniotic fluid specimens submitted to Colombia's National Institute of Health as part of national Zika virus surveillance from January 2016 to January 2017. Specimens (maternal serum, amniotic fluid, cord blood, umbilical cord tissue, and placental tissue) were tested for the presence of Zika virus RNA with the use of a singleplex or multiplex real-time reverse transcriptase-polymerase chain reaction assay. Birth defect information was abstracted from maternal prenatal and infant birth records and reviewed by expert clinicians. Chi-square and Fisher's exact tests were used to compare the frequency of Zika-associated birth defects (defined as brain abnormalities [with or without microcephaly, but excluding neural tube defects and their associated findings] or eye abnormalities) by frequency of detection of Zika virus RNA in amniotic fluid. Our analysis included 128 women with amniotic fluid specimens. Seventy-five women (58%) had prenatally collected amniotic fluid; 42 women (33%) had amniotic fluid collected at delivery, and 11 women (9%) had missing collection dates. Ninety-one women had both amniotic fluid and other clinical specimens submitted for testing, which allowed for comparison across specimen types. Of those 91 women, 68 had evidence of Zika virus infection based on detection of Zika virus RNA in ≥1 specimen. Testing of amniotic fluid that was collected prenatally or at delivery identified 39 of these Zika virus infections (57%; 15 [22%] infections were identified only in amniotic fluid), and 29 infections (43%) were identified in other specimen types and not amniotic fluid. Among women who were included in the analysis, 89 had pregnancy outcome information available, which allowed for the assessment of the presence of Zika-associated birth defects. Zika-associated birth defects were significantly (P<.05) more common among pregnancies with Zika virus RNA detected in amniotic fluid specimens collected prenatally (19/32 specimens; 59%) than for those with no laboratory evidence of Zika virus infection in any specimen (6/23 specimens; 26%), but the proportion was similar in pregnancies with only Zika virus RNA detected in specimens other than amniotic fluid (10/23 specimens; 43%). Although Zika-associated birth defects were more common among women with any Zika virus RNA detected in amniotic fluid specimens (ie, collected prenatally or at delivery; 21/43 specimens; 49%) than those with no laboratory evidence of Zika virus infection (6/23 specimens; 26%), this comparison did not reach statistical significance (P=.07). Testing of amniotic fluid provided additional evidence for maternal diagnosis of Zika virus infection. Zika-associated birth defects were more common among women with Zika virus RNA that was detected in prenatal amniotic fluid specimens than women with no laboratory evidence of Zika virus infection, but similar to women with Zika virus RNA detected in other, nonamniotic fluid specimen types.

Development of multiplex real-time reverse-transcriptase polymerase chain reaction assay for simultaneous detection of Zika, dengue, yellow fever, and chikungunya viruses in a single tube.

Zika virus (ZIKV), dengue virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) share the same mosquito vectors and have similar clinical manifestations early stage of infection. Therefore, simultaneously differentiating these viruses from each other is necessary. We developed a multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the differentiation of these four viruses in a single tube. The linear range was established by regression analysis, and the R2 value for each viruswas ≥0.98, and the 95% lower limit of detectionfor each virus was as follows (copies/reaction): ZIKV-Asian, 9; ZIKV-Africa, 15; CHIKV, 11; DENV-1, 19; DENV-2, 13; DENV-3, 24; DENV-4, 36; and YFV, 17. Meanwhile, our multiplex real-time RT-PCR has a good consistency with the commercial singleplex assay. In summary, the developed assay can be effectively used for the diagnosis of ZIKV, DENV, CHIKV, and YFV infections.

Investigation of avian influenza infection in wild birds in Ismailia and Damietta cities, Egypt.

Aim:This study was carried out to monitor avian influenza (AI) infection in wild birds in Egypt.Materials and Methods:A total of 135 wild birds were examined for the presence of H5, H7, and H9 hemagglutination inhibition antibodies. Organs and swab samples of 75 birds were screened by multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) to detect AI subtypes H5, H7, and H9 matrix genes.Results:The highest seropositive result was recorded in cattle egrets (90.9%) followed by crows (88.6%), semi-captive pigeons (44.8%), and moorhens (39.1%). In cattle egrets, semi-captive pigeons and moorhens, H5 antibodies predominated. In crows, H9 antibodies predominated. Multiple infections with two or three virus subtypes were highest in crows (6/39, 15.4%) followed by cattle egrets (3/30, 10%) and moorhens’ (1/9, 11.1%) positive samples. Multiplex RRT-PCR results revealed two positive samples in cattle egrets and moorhens.Conclusion:The results indicated high seropositive rates against AI virus subtypes H5 and H9 in the examined wild birds. Multiple infections with more than one AI virus (AIV) subtypes were detected in some birds. This requires a collaboration of efforts to monitor AIV infection in wild birds and implement suitable early intervention measures.

Clinical and Laboratory Profile of Dengue Viral Infections in and around Mangalore, India

Background: Dengue (DEN) is being recognised as the world’s major emerging tropical disease. Clinically, DEN may resemble other infections such as malaria, leptospirosis, and typhoid, and thus, laboratory investigations are required for definitive diagnosis. Secondary DEN infection, caused most often by dengue virus (DENV) serotypes 2 and 3, is known to present with severe disease manifestations. This study was undertaken to examine the clinical and laboratory profile of DEN viral infections and to determine the circulating serotypes in and around Mangalore, India. Materials and Methods: Serum samples from 285 clinically suspected cases of DEN in and around Mangalore between September 2013 and January 2014 were processed for detection of DEN IgM and IgG antibodies and nonstructural 1 (NS1) antigen using commercial ELISA kits. Detection of DEN viral RNA and serotyping was done by multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The clinical and haematological profiles of the patients were analysed. Results: Serum samples from 83 (29%) patients were positive for DEN NS1 antigen and/or IgM antibodies. 33 (45%) out of 73 serum samples processed by multiplex real-time RT-PCR were positive for DEN viral RNA. DEN-1, -2 and -3 were the serotypes identified in this study. Fever was the most common presenting symptom followed by myalgia/arthralgia. Majority of the patients had thrombocytopaenia. Conclusion: Early detection of DEN can be achieved effectively using NS1 ELISA and IgM capture ELISA. Circulating DENV serotypes should be closely monitored for prevention of fatal outcomes in secondary infections.

Clinical Epidemiology of Bocavirus, Rhinovirus, Two Polyomaviruses and Four Coronaviruses in HIV-Infected and HIV-Uninfected South African Children

BackgroundAdvances in molecular diagnostics have implicated newly-discovered respiratory viruses in the pathogenesis of pneumonia. We aimed to determine the prevalence and clinical characteristics of human bocavirus (hBoV), human rhinovirus (hRV), polyomavirus-WU (WUPyV) and –KI (KIPyV) and human coronaviruses (CoV)-OC43, -NL63, -HKU1 and -229E among children hospitalized with lower respiratory tract infections (LRTI).MethodsMultiplex real-time reverse-transcriptase polymerase chain reaction was undertaken on archived nasopharyngeal aspirates from HIV-infected and –uninfected children (<2 years age) hospitalized for LRTI, who had been previously investigated for respiratory syncytial virus, human metapneumovirus, parainfluenza I–III, adenovirus and influenza A/B.ResultsAt least one of these viruses were identified in 274 (53.0%) of 517 and in 509 (54.0%) of 943 LRTI-episodes in HIV-infected and -uninfected children, respectively. Human rhinovirus was the most prevalent in HIV-infected (31.7%) and –uninfected children (32.0%), followed by CoV-OC43 (12.2%) and hBoV (9.5%) in HIV-infected; and by hBoV (13.3%) and WUPyV (11.9%) in HIV-uninfected children. Polyomavirus-KI (8.9% vs. 4.8%; p = 0.002) and CoV-OC43 (12.2% vs. 3.6%; p<0.001) were more prevalent in HIV-infected than –uninfected children. Combined with previously-tested viruses, respiratory viruses were identified in 60.9% of HIV-infected and 78.3% of HIV-uninfected children. The newly tested viruses were detected at high frequency in association with other respiratory viruses, including previously-investigated viruses (22.8% in HIV-infected and 28.5% in HIV–uninfected children).ConclusionsWe established that combined with previously-investigated viruses, at least one respiratory virus was identified in the majority of HIV-infected and HIV-uninfected children hospitalized for LRTI. The high frequency of viral co-infections illustrates the complexities in attributing causality to specific viruses in the aetiology of LRTI and may indicate a synergetic role of viral co-infections in the pathogenesis of childhood LRTI.

The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3'-end primer region on real-time PCR using the 5'-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A-C, C-A, T-G, G-T) to severe impact (>7.0 cycle threshold, eg, A-A, G-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.

Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A

Avian influenza viruses are pathogens of economical and public health concerns. However, infections caused by low pathogenic avian influenza particularly H9N2 subtype are not associated with clear clinical features. Hence, rapid detection and subtyping of the virus will enable immediate measures to be implemented for preventing widespread transmission. This study highlights the development of a multiplex real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay using SYBR Green 1 chemistry for universal detection of avian influenza viruses and specific subtyping of H9N2 isolates based on melting temperatures ( T m) discriminations. Three melting peaks generated simultaneously at temperatures 85.2 ± 1.0, 81.9 ± 0.9 and 78.7 ± 0.9 °C represent NP, H9 and N2 gene products, respectively. The RRT-PCR assay was about 10–100-fold more sensitive when compared to the conventional RT-PCR method using reference H9N2 isolate. In addition, the RRT-PCR assay was 100% sensitive as well as 92% specific according to the standard virus isolation method in detecting experimentally infected specific-pathogen-free (SPF) chickens.

A one-step multiplex real-time RT-PCR for detection and typing of bovine viral diarrhea viruses

A one-step multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SmartCycler technology and TaqMan probes was developed for detection and typing of bovine viral diarrhea viruses (BVDV). Common primers and type-specific (BVDV1 and BVDV2) TaqMan probes were designed in the 5′-untranslated region of the viral genome. The real-time assay was able to detect 10–100 TCID 50 of virus, with correlation coefficient ( r 2) values of 0.998 and 0.999 for BVDV1 and BVDV2, respectively. The assay accurately typed 54 BVDV strains and field isolates and specificity of the TaqMan probes was further demonstrated by the lack of reactivity with the closely related Pestiviruses, classical swine fever virus and border disease virus. The assay was also shown to have high reproducibility. When the assay was compared with virus isolation for bovine serum samples, there was full agreement between the tests. Thus, the one-step real-time RT-PCR assay appears to be a rapid, sensitive, and specific test for detection and typing of BVDV.

TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type

Chronic myelogenous leukemia is characterized by the presence of the reciprocal t(9;22)(q34;q11) in which c-abl located on chromosome 9, and the bcr locus located on chromosome 22, are disrupted and translocated creating a novel bcr-abl fusion gene residing on the derivative chromosome 22. In most cases, the breakpoint in abl occurs within intron 1. Depending on the breakpoint in bcr, exon 2 of abl (a2) joins with exons 1 (e1), 13 (b2), or 14 (b3), or rarely to exon 19 (e19) of bcr resulting in chimeric proteins of p190, p210 and p230, respectively. Currently, several multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays for detecting bcr-abl are available to assess the levels of the three common fusion transcripts, b2a2, b3a2 and e1a2. Although these assays circumvent the requirement for individual fusion sequence quantitative polymerase chain reaction-based assays, they do not identify the specific fusion transcript. Knowledge of the latter is useful to rule out false-positive results and to compare clones before and after therapy. We designed a novel multiplex real-time RT-PCR assay to detect bcr-abl that allows accurate quantification and determination of the specific fusion transcript. In this assay, abl primer labeled at its 5′ end with the fluorescent dye NED (Applied Biosystems) is incorporated into the bcr-abl fusion product during amplification. The NED fluorescent dye in abl primer, without interfering with fluorescent TaqMan probe signal, allows subsequent identification of the fusion transcript by semiautomated high-resolution capillary electrophoresis and GeneScan analysis.

Development of multiplex real-time RT-PCR as a diagnostic tool for avian influenza.

A multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) assay for the simultaneous detection of the H5 and H7 avian influenza hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) reporter dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro transcribed RNA templates, to have a reproducible detection limit for H7 of approximately 10(4) HA gene copies and approximately 10(4)-10(5) HA gene copies of H5. A direct comparison of H5-H7 multiplex RRT-PCR with hemagglutination inhibition (HI) was performed with 83 AI RRT-PCR and virus isolation positive tracheal and cloacal swab samples obtained from various avian species and environmental swabs from live-bird markets in New York and New Jersey. Both multiplex RRT-PCR and HI agreed on the subtype determination of 79 (95.2%) of the 83 samples, of which 77 were positive for H7 and two were determined to be non-H5/non-H7 subtypes. No samples were determined to be the H5 subtype by either assay.

Influence of Preassay and Sequence Variations on Viral Load Determination by a Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction for Feline Immunodeficiency Virus

Determination of retroviral load is an important tool in the investigation of the success of therapeutic or vaccination trials in patients infected with lentiviruses such as HIV, or with their simian (SIV) or feline (FIV) counterparts. We have developed an one-tube quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay based on the ABI Prism 7700 Sequence Detection System (TaqMan) to quantify the viral load of FIV-infected cats. Two different primer/probe systems were designed to detect a broad range of clade A FIV isolates. Both systems are characterized by excellent reproducibility, high sensitivity, and a wide range of quantification. As a consequence of this improved precision in the quantitative RT-PCR, preassay variations have greater impact on the accuracy of the viral load estimation. To compensate for these variations, we improved the assay and developed a multiplex real-time RT-PCR, which allows simultaneous calculation of the viral copy number and the individual recovery rate in an one-tube reaction. This enables the rapid and accurate calculation of copy number independent of preassay variations. In further studies, two additional real-time RT-PCR assays were designed and used to investigate the influence of sequence variations in the binding regions for either the primers or probe. Sequence mismatches in this region had a significant effect (up to 4 logarithmic decades) on reaction efficiency. In view of the inherent variability of retroviral sequences, these results underline the necessity to check reaction efficiencies before determining viral load.

Influence of Preassay and Sequence Variations on Viral Load Determination by a Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction for Feline Immunodeficiency Virus

Influence of Preassay and Sequence Variations on Viral Load Determination by a Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction for Feline Immunodeficiency Virus