Multiplex Reverse Transcriptase PCR Research Articles

One-step endpoint RT-PCR assays for confirmatory body fluid identification.

Messenger RNA (mRNA) expression analysis is increasingly used in casework, in the form of multiplex two-step reverse transcriptase PCR (RT-PCR) assays such as CellTyper 2 (CT2), developed by the Institute of Environmental Science and Research (ESR). This paper presents the development of a one-step endpoint RT-PCR workflow to improve the efficiency and precision of confirmatory body fluid identification. A comparative study of commercial one-step RT-PCR kits was undertaken, with the highest performing kit (RNA to CT) retained for further development. Sensitivity, specificity across body fluids, and precision was assessed simultaneously using receiver operating characteristic (ROC) curves. An optimal RFU cut-off value which maximised sensitivity and specificity was determined for each marker. All assays performed significantly better when compared to the equivalent of a completely uninformative test (area under the curve of 0.5) for their target body fluid. Sensitivity varied between different donors, but the limit of detectionss were estimated as follows; saliva markers HTN3: 1 in 100 dilution of a whole buccal swab and FDCSP: 1 in 10 dilution of a whole buccal swab, circulatory blood marker SLC4A1: 0.1µL blood, menstrual fluid markers STC1, MMP10: 1 in 10 dilution of a whole menstrual swab, spermatozoa markers PRM1, TNP1: 0.1µL semen, seminal fluid markers KLK2: 0.1µL semen and MSMB: 0.01µL semen, and vaginal material marker CYP2B7P: 1 in 1000 dilution of a whole vaginal swab. The method successfully detected most body fluids in a range of simple mixtures with 77 out of 80 markers observed when expected. The developed one-step endpoint RT-PCR assays lack the sensitivity and precision required for forensic casework and provide little benefit when compared with standard two-step endpoint RT-PCR, other than minimal time and cost savings, similar sensitivity, and improved precision for some markers. As both methods utilise endpoint RT-PCR, they have the same narrow linear dynamic range. The novel method is therefore similarly susceptible to varied RNA input, a major disadvantage of this approach. The limited sensitivity and precision consistently encountered with endpoint RT-PCR - regardless of cDNA synthesis strategy - could be addressed by a real-time PCR approach.

Diagnostic Performance and Tolerability of Saliva and Nasopharyngeal Swab Specimens in the Detection of SARS-CoV-2 by RT-PCR.

Saliva is a promising alternative for a nasopharyngeal swab (NPS) in specimen collection to detect SARS-CoV-2. We compared the diagnostic performance and tolerability of saliva collection versus NPS in a clinical setting. Paired NPS and saliva specimens were collected sequentially from participants (n = 250) at the Turku University Hospital drive-in coronavirus testing station in the spring of 2022, with Omicron BA.2 as the dominant SARS-CoV-2 variant. Discomfort and preference for the sampling method were assessed. The specimens were analyzed for SARS-CoV-2 using real-time multiplex reverse transcriptase PCR (RT-PCR) with a laboratory-developed test (LDT) and two commercial kits (PerkinElmer SARS-CoV-2 and PerkinElmer SARS-CoV-2 Plus) for several target genes. Among the 250 participants, 246 had respiratory symptoms. With LDT, SARS-CoV-2 was detected in 135 and 134 participants from NPS and saliva, respectively. Of the 250 specimens, 11 gave a discordant outcome, resulting in excellent agreement between the specimen types (Cohen's kappa coefficient of 0.911; P = 0.763). The cycle threshold (CT) values of LDT and commercial kit target genes were significantly lower from NPS than from saliva. A total of 172 (69%) participants assessed saliva sampling as more tolerable than NPS (P < 0.0001). Our findings present saliva as an applicable alternative for SARS-CoV-2 diagnostics. However, the lower CT values obtained from NPS indicate that NPS may be a slightly more sensitive specimen type. Participants preferred saliva sampling, although delivering an adequate volume of saliva was challenging for some participants. IMPORTANCE The extensive testing of SARS-CoV-2 is vital in controlling the spread of COVID-19. The reference standard for specimen collection is a nasopharyngeal swab (NPS). However, the discomfort of NPS sampling, the risk of nosocomial infections, and global material shortages have accelerated the development of alternative testing methods. Our study demonstrates that patients tolerate saliva sampling better than NPS. Of importance, although the RT-PCR qualitative test results seem to correspond between NPS and saliva, we show significantly lower CT values for NPS, compared to saliva, thus contradicting the suggested superiority of the saliva specimen over NPS in the detection of the Omicron variants of SARS-CoV-2. Future research is still required to enable individual planning for specimen collection and to determine the effects of different SARS-CoV-2 variants on the sensitivity of the saliva matrix.

Assessment of a multiplex RT-PCR for Simultaneous, Rapid Screening of Common Viral Infections of Central Nervous System: A Prospective Study for Enteroviruses and Herpesviruses

Abstract Introduction Acute meningitis is a common neurological disorder that affects both children and adults and has a high mortality rate. This study aimed to create a multiplex reverse transcriptase PCR system for screening clinical samples for the presence of the two viruses currently considered to be the most common causes of acute meningitis in Asia. Materials and Methods A single-tube RT multiplex PCR assay was developed and tested for sensitivity and specificity using primers that have been commonly used to screen for herpes simplex viruses 1 and 2 (HSV-1/2) and enterovirus (EV) in clinical samples. The procedure was then used to screen 303 clinical samples for the target viruses, which included 101 feces samples, 101 throat swabs, and 101 cerebrospinal fluid (CSF) samples obtained from 101 hospitalized Iranian children with suspected viral meningitis/meningoencephalitis, and the findings were compared to those of an RT monoplex PCR method. Results The RT-PCR approach demonstrated high precision, with no non-target virus amplification. The results of using this assay to screen clinical samples revealed that RT monoplex PCR had the same sensitivity as RT multiplex PCR for the three different types of specimens. Conclusions This newly developed multiplex RT-PCR method is a simple, fast diagnostic tool that can be used to screen clinical samples for viruses that cause acute meningitis/meningoencephalitis in children.

Diagnostic Validation of a Clinical Laboratory-Oriented Targeted RNA Sequencing System for Detecting Gene Fusions in Hematologic Malignancies

Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest with a high resolution and throughput. However, RNA-seq has not been widely performed in clinical molecular laboratories because of the complexity of data processing and interpretation. We developed and validated a customized RNA-seq panel and data processing protocol for fusion detection using 4 analytical validation samples and 51 clinical samples, covering seven types of hematologic malignancies. Analytical validation showed that the results for target gene coverage and between- and within-run precision and linearity tests were reliable. Using clinical samples, RNA-seq based on filtering and prioritization strategies detected all 25 known fusions previously found by multiplex reverse transcriptase-PCR and fluorescence in situ hybridization. It also detected nine novel fusions. Known fusions detected by RNA-seq included two IGH rearrangements supported by expression analysis. Novel fusions included six that targeted just one partner gene. In addition, 18 disease- and drug resistance-associated transcript variants in ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were designated simultaneously. Expression analysis showed distinct clustering according to subtype and lineage. In conclusion, this study showed that our customized RNA-seq system had a reliable and stable performance for fusion detection, with enhanced diagnostic yield for hematologic malignancies in a clinical diagnostic setting.

Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase PCR (RT-qPCR) to detect three nonsynonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2-positive specimens from our San Francisco Bay Area population. Between 1 December 2020 and 1 March 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K plus N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing of a validation subset of 229 specimens and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed the rapid emergence of the L452R variant in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

Co-distribution of dengue and Chikungunya viruses in Aedes mosquitoes of Delhi, India.

In India, vector-borne diseases, dengue and chikungunya are major public health concerns. In recent decades, dengue outbreaks have been reported in almost every part of India. In 2016, India recorded 101388 dengue cases and 210 deaths, including 4337 cases and six deaths in Delhi, whereas Chikungunya outbreaks were reported from several states in 2006, with 1.3 million cases. The Dengue virus (DENV) and Chikungunya virus (CHIKV) are both transmitted by the same Aedes mosquito species. DENV and CHIKV co-infections have been reported in 13 of 98 countries, with both viruses being transmitted locally. The reasons for the sudden upsurge in cases of these diseases are undetermined. From March to December 2016, a study was carried out in 66 localities of Delhi in collaboration with the Municipal Corporation of Delhi. Localities were selected on the basis of confirmed dengue cases reported during the last five years and the study area was visited once a month. A door-to-door entomological survey was conducted to identify Aedes breeding in all water-filled containers in and around houses. Both immature and mature stages of Aedes mosquitoes were collected. Mosquitoes were pooled (n≤10 each for male and female) breeding site-wise and stored in Trizol at -80°C. The Chikungunya and dengue viruses were detected using a multiplex RT-PCR. A total of 981 Aedes mosquitos were distributed among 146 Pools, and DENV and CHIKV were detected using Multiplex Reverse Transcriptase-PCR. Chikungunya virus was identified in 19 pools of females captured adults, whereas dengue virus was found in 8 pools of females captured adults. There was no evidence of coinfection in any of the pools. In endemic areas, continuous surveillance for both dengue and Chikungunya viruses is required to identify and characterize these viral pathogens. This information will also help implement effective strategies to combat outbreaks produced by these emerging viral pathogens.

BCR-ABL Gene Transcript Types of Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia.

Background:The aim of this study was analyzing the BCR-ABL transcript types of patients with chronic myeloid leukemia (CML) in Dr Sardjito General Hospital, Yogyakarta, Indonesia. This study is very relevant because the data concerning BCR-ABL gene transcript types is very limited in Indonesia. Furthermore, it is important for patient’s management, particularly in defining the tyrosine kinase inhibitors (TKIs) therapy and monitoring after therapy. The introduction of TKIs has become a major advance in the management of patients with CML, especially in the chronic phase (CML-CP), in which most patients are diagnosed. Methods:One hundred eighty five (185) of 370 recruited patients were included in this study (2010–2014). RNA samples were isolated from mononuclear cells of peripheral blood of the subjects taken at primary diagnosis. Detection of BCR-ABL gene transcript types was done using multiplex reverse transcriptase PCR (multiplex RT-PCR) and/or nested PCR following the cDNA synthesis. When the first PCR set failed to amplify the BCR-ABL gene, RT-conventional PCR and/or nested PCR would be applied. The proportion of each transcript type was calculated among the BCR-ABL positive CML patients. Results:Approximately 99% (183/185) of CML patients are BCR-ABL positive, with the most common type is major b3a2 (136/183; 74.3%), followed by major b2a2 (41/183; 22.4%). Two samples (1.1%) showed co-expression of b3a2 and b2a2; 1 sample showed co-expression of b3a2 and fragment at 500bp; and 3 samples showed uncommon fragments. Conclusion:Ninety nine percent (99%) of CML patients in Yogyakarta, Indonesia are BCR-ABL positive, with 74.3% have b3a2 transcript, 22.4% have b2a2 trascript, 1.1% have co-expression of b3a2 and b2a2 transcript, and the rest (2.2%) have uncommon bands that still need to be confirmed.

Advances in laboratory assays for detecting human metapneumovirus.

Human metapneumovirus (HMPV) is one of the major causes of acute respiratory tract infection (ARI) and shows high morbidity and mortality, particularly in children and immunocompromised patients. Various methods for detecting HMPV have been developed and applied in clinical laboratories. When reviewing the literature, we found that polymerase chain reaction (PCR)-based assays have been most frequently and consistently used to detect HMPV. The most commonly used method was multiplex reverse transcriptase-PCR (RT-PCR; 57.4%), followed by real-time RT-PCR (38.3%). Multiplex RT-PCR became the more popular method in 2011–2019 (69.7%), in contrast to 2001–2009 (28.6%). The advent of multiplex PCR in detecting broader viral pathogens in one run and coinfected viruses influenced the change in user preference. Further, newly developed microarray technologies and ionization mass spectrometry were introduced in 2011–2019. Viral culture (including shell vial assays) and fluorescent immunoassays (with or without culture) were once the mainstays. However, the percentage of studies employing culture and fluorescent immunoassays decreased from 21.4% in 2001–2010 to 15.2% in 2011–2019. Meanwhile, the use of PCR-based methods of HMPV detection increased from 78.6% in 2001–2010 to 84.8% in 2011–2019. The increase in PCR-based methods might have occurred because PCR methods demonstrated better diagnostic performance, shorter hands-on and run times, less hazards to laboratory personnel, and more reliable results than traditional methods. When using these assays, it is important to acquire a comprehensive understanding of the principles, advantages, disadvantages, and precautions for data interpretation. In the future, the combination of nanotechnology and advanced genetic platforms such as next-generation sequencing will benefit patients with HMPV infection by facilitating efficient therapeutic intervention. Analytical and clinical validation are required before using new techniques in clinical laboratories.

Combining multiplex PCR and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals

The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.

Epidemiology and genetic diversity of group A rotavirus in acute diarrhea patients in pre-vaccination era in Himachal Pradesh, India

Acute gastroenteritis due to Group A rotaviruses remains the leading cause of mortality and morbidity in children in developing countries. India introduced its indigenous rotavirus vaccine Rotavac® in 2016 and Himachal Pradesh (HP) the first state to launch it. The present study aimed to evaluate rotavirus strain diversity associated with AGE prior to vaccine introduction in HP. A total of 331 fecal specimens collected from diarrheic children hospitalized at RPGMC Tanda, HP between July-2014 and June-2016 were screened for RVA by EIA. Rotavirus RNA was extracted by TRIZOL method and analyzed by RNA-PAGE. G/P typing was performed using semi-nested multiplex reverse transcriptase PCR. Rotavirus was detected in 45% (n = 149/331) of diarrheic children, with highest rate observed in the 6–11 months age group (47%). Vomiting was found more frequently associated with RV-infection. Among G-types, G12 was found most prevalent (33.1%) followed by G1 (28.4%), G9 (12.2%), G2 (9.5%), G3 (3.4%) and G10 (2.7%). G4 (0.7%) strains were rarely detected. Among P-types, P[6] was the most prevalent (40.5%) followed by P[8] (29.1%) and P[4] (14.2%). Of note, genotypes G3 and P[11] were detected for the first time in HP. Among G/P combinations, G12P[6] was most prevalent (30.4%) followed by G1P[8] (20.3%), G2P[4] (4.7%), G1P[6] (3.4%) and G3P[8] (2.7%). Interestingly, our study observed high percentage of unusual strains (14.2%) namely G9P[4], G2P[6], G2P[8], G12P[4] and G1P[11]. The regionally common strains G3P[6], G4P[6], G9P[6], G9P[8], G10P[6], G10P[8] and G12P[8] strains were very rarely detected. Of interest, RNA migration pattern of G1P[8] was DS-1 like and genomic heterogeneity was observed within G12P[4] strains with both long and short electropherotypes. Our study highlights rich genetic diversity with emergence of rare rotavirus strains circulating in HP and provides baseline data prior to Rotavac® introduction that will help to gauge the impact of the Rotavac® vaccine in HP.

Real-time quantitative PCR: a reliable molecular diagnostic and follow-up tool for 'minimal residual disease' assessment in chronic myeloid leukemia.

Molecular monitoring of BCR-ABL transcript levels by real-time quantitative PCR is increasingly being used to diagnose the disease and assess treatment response in patients with chronic myeloid leukemia (CML). This has become particularly relevant when residual levels of leukemia usually fall below the level of detection by cytogenetic analysis. Forty-two CML patients, including 18 males (42.86%) and 24 females (57.14%) aged 7–75 years, were enlisted for the study and followed-up for the response to imatinib treatment. Patients were subjected to Multiplex RT-PCR (reverse-transcriptase PCR) and were all found to harbor either e13a2 or the e14a2, which could be analyzed by a single Taqman probe based quantitation kit (Geno-Sen’s) to quantitate the BCR-ABL transcript load. The Multiplex RT-PCR and peripheral blood cytogenetics providing specific and sensitive detection of BCR-ABL fusion transcripts and metaphase signal load respectively were used as parallel reference tools to authenticate the q-PCR findings. There was 100% concordance between the multiplex RT-PCR and the q-PCR as every positive RT-PCR assay for a transcript reflected as q-PCR load of above 0% for that transcript. q-PCR also demonstrated a strong Pearson correlation with the cytogenetic response.

Impact of respiratory viruses in the neonatal intensive care unit.

ObjectiveTo describe the epidemiology and clinical impact of respiratory viruses in a neonatal intensive care unit (NICU).Study designWe conducted a retrospective observational study of infants with respiratory viruses detected by multiplex reverse-transcriptase PCR from May 2012 to May 2017. The proportion of symptomatic vs. asymptomatic infants and associated morbidity were assessed. The association of infection prevention and control (IP&C) strategies and transmission was ascertained.ResultsRespiratory viruses were detected in 83 infants representing 86 unique episodes during which infants remained asymptomatic in 15 (17%). Of the 71 symptomatic episodes, only 45% were associated with increased respiratory and/or nutritional support. Rhinovirus/enteroviruses were most common (69%) and involved nine of 12 transmission events. IP&C strategies including restricting visitors <12 years of age and screening exposed infants were associated with decreased transmission rates.ConclusionsNICU patients can be asymptomatic carriers of respiratory viruses. Identification of such infants is important to prevent transmission in the NICU.

Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus

Nine influenza virus neuraminidase (NA) subtypes have been identified in poultry and wild birds. Few methods are available for rapid and simple NA subtyping. Here we developed a multiplex probe combination-based one-step real-time reverse transcriptase PCR (rRT-PCR) to detect nine avian influenza virus NA subtypes. Nine primer-probe pairs were assigned to three groups based on the different fluorescent dyes of the probes (FAM, HEX, or Texas Red). Each probe detected only one NA subtype, without cross reactivity. The detection limit was less than 100 EID50 or 100 copies of cDNA per reaction. Data obtained using this method with allantoic fluid samples isolated from live bird markets and H9N2-infected chickens correlated well with data obtained using virus isolation and sequencing, but was more sensitive. This new method provides a specific and sensitive alternative to conventional NA-subtyping methods.

Differential protein stability and clinical responses ofEML4-ALK fusion variants to various ALK inhibitors in advancedALK-rearranged non-small cell lung cancer

Anaplastic lymphoma kinase (ALK) inhibition using crizotinib has become the standard of care in advanced ALK-rearranged non-small cell lung cancer (NSCLC), but the treatment outcomes and duration of response vary widely. Echinoderm microtubule-associated protein-like 4 (EML4)-ALK is the most common translocation, and the fusion variants show different sensitivity to crizotinib in vitro. However, there are only limited data on the specific EML4-ALK variants and clinical responses of patients to various ALK inhibitors. By multiplex reverse-transcriptase PCR, which detects 12 variants of known EML4-ALK rearrangements, we retrospectively determined ALK fusion variants in 54 advanced ALK rearrangement-positive NSCLCs. We subdivided the patients into two groups (variants 1/2/others and variants 3a/b) by protein stability and evaluated correlations of the variant status with clinical responses to crizotinib, alectinib, or ceritinib. Moreover, we established the EML4-ALK variant-expressing system and analyzed patterns of sensitivity of the variants to ALK inhibitors. Of the 54 tumors analyzed, EML4-ALK variants 3a/b (44.4%) was the most common type, followed by variants 1 (33.3%) and 2 (11.1%). The 2-year progression-free survival (PFS) rate was 76.0% [95% confidence interval (CI) 56.8-100] in group EML4-ALK variants 1/2/others versus 26.4% (95% CI 10.5-66.6) in group variants 3a/b (P = 0.034) among crizotinib-treated patients. Meanwhile, the 2-year PFS rate was 69.0% (95% CI 49.9-95.4) in group variants 1/2/others versus 32.7% (95% CI 15.6-68.4) in group variants 3a/b (P = 0.108) among all crizotinib-, alectinib-, and ceritinib-treated patients. Variant 3a- or 5a-harboring cells were resistant to ALK inhibitors with >10-fold higher half maximal inhibitory concentration in vitro. Our findings show that group EML4-ALK variants 3a/b may be a major source of ALK inhibitor resistance in the clinic. The variant-specific genotype of the EML4-ALK fusion allows for more precise stratification of patients with advanced NSCLC.

Beyond just being foot soldiers – osmotin like protein (OLP) and chitinase (Chi11) genes act as sentinels to confront salt, drought, and fungal stress tolerance in tomato

Osmotin like protein (OLP) and chitinase (Chi11) belong to pathogenesis-related (PR) class of proteins and are induced during both biotic and abiotic stresses. Transgenic tomato was developed with OLP and Chi11 via in vitro and in planta transformation methods. Transgenes integration and transcript levels were confirmed by multiplex PCR, DNA blot, and multiplex reverse transcriptase PCR. Homozygous T2 transgenics when evaluated for salt, drought, and fungal stresses showed enhanced tolerance compared to untransformed controls (UC). Transgenics showed enhanced endochitinase activity and root biomass under normal conditions. Transformants also displayed higher proline content, K+, relative water content, chlorophyll fluorescence, total biomass, vascular conductivity, and fruit yield than the UC under stress conditions. Co-immunoprecipitation revealed that Chi11 co-expresses with phosphofructokinase2 (PFK2), which may play a role in enhanced root biomass. qPCR analysis resulted in higher transcript levels of OLP, Chi11, and PFK2 in transgenics as compared to the untransformed controls. Our findings suggest the use of multiples genes to confer multiple stress tolerance for enhanced crop productivity. This work unveils a new molecular player PFK2, which may play a role in enhanced root biomass.

The added values of multiplex reverse transcriptase‐PCR followed by mutation screening in the initial evaluation of acute leukemia

This study investigates the benefits of using multiplex reverse transcriptase-PCR (RT-PCR) in addition to standard karyotyping during the initial evaluation of acute leukemia. A total of 1114 consecutive specimens from patients with acute leukemia were tested using a commercial multiplex RT-PCR kit (HemaVision, DNA Diagnostic). NPM1 and CEBPA mutations were selectively tested in acute myeloid leukemia (AML) patients with multiplex RT-PCR negativity. In specimens with optimal cytogenetics, the frequency of recurrent translocations was 31.3%, and cryptic translocations were detected in 2.1% of samples. The concordance rate between karyotyping and multiplex RT-PCR was 97.5%. In addition to the established functions, we demonstrated the additional benefits of multiplex RT-PCR, including successful molecular characterization, even in cytogenetically suboptimal specimens (5.7%); detection of submicroscopic aberrations (1.0%); detection of rare but potentially significant translocations or variants (2.5%); selection of AML candidates for mutation analysis (68.3%); and finally exclusion of recurrent translocations in patients with acute lymphoblastic leukemia or mixed phenotype acute leukemia (22.5%). We reconfirmed the accuracy and reliability of multiplex RT-PCR for diagnosing acute leukemia and demonstrated additional advantages of this system for the initial evaluation of acute leukemia. Thus, multiplex RT-PCR is worth considering in diagnostic testing of acute leukemias.

Single-cell analysis of glandular T cell receptors in Sjögren's syndrome.

CD4+ T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren's syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4+CD45RA- T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies.

Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan.

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase–PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/μl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

Multiplex PCR and Microarray for Detection of Swine Respiratory Pathogens.

SummaryPorcine respiratory disease complex (PRDC) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. No simple, single‐reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with PRDC. Furthermore, the detection of most of the bacterial pathogens implicated in PRDC currently requires time‐consuming culture‐based methods that can take several days to obtain results. In this study, a novel prototype automated microarray that integrates and automates all steps of post‐PCR microarray processing for the simultaneous detection and typing of eight bacteria and viruses commonly associated with PRDC is described along with associated multiplex reverse transcriptase PCR. The user‐friendly assay detected and differentiated between four viruses [porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus, porcine circovirus type 2, porcine respiratory corona virus], four bacteria (Mycoplasma hyopneumoniae, Pasteurella multocida, Salmonella enterica serovar Choleraesuis, Streptococcus suis), and further differentiated between type 1 and type 2 PRRSV as well as toxigenic and non‐toxigenic P. multocida. The assay accurately identified and typed a panel of 34 strains representing the eight targeted pathogens and was negative when tested with 34 relevant and/or closely related non‐target bacterial and viral species. All targets were also identified singly or in combination in a panel of clinical lung samples and/or experimentally inoculated biological material.