Duplex qPCR Assay Research Articles

Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples

Verticillium wilt, Verticillium stem striping, and Verticillium stripe are common disease names that all denote infection caused by Verticillium longisporum on canola or other brassica crops. In this study, a quantitative PCR (qPCR) and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level across all whole-genome sequenced strains. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-µl reaction or 700 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans, to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Development of a qPCR Duplex Assay for simultaneous detection of Fascioloides magna and Galba truncatula in eDNA samples: Monitoring beyond boundaries

Parasites constitute a significant economic burden and highly impact environmental, public, and animal health. The emergence of many parasitic diseases is environmentally mediated and they share the same biogeography with humans and both domestic and wild animals. American liver fluke, Fascioloides magna – a trematode parasite of domestic and wild ungulates – is an example of the anthropogenic introduction of an “invasive alien species” in Italy and Europe. Multiple introductions to Europe have led to the biogeographical expansion of the parasite across the Danube region mainly provided by the presence of suitable habitats for all hosts involved in the parasite's life cycle, human-assisted transport, and drastic environmental events such as flooding. In Italy, it was introduced and established in La Mandria Regional Park (LMRP) near Turin in 1865 along with imported wapitis (Cervus elaphus canadensis) from North America (Bassi, 1875), but with no reported expansion to the surrounding areas. LMRP isolated F. magna focus, poses an important threat of possible expansion since the enclosed area is vulnerable to occasional bidirectional passage of roe deer. Additionally, tributary rivers to the Po river system, traversing the enclosed area, could further bolster the possibility of such spread. In this study, we developed a duplex qPCR assay for F. magna and its principal intermediate host Galba truncatula optimized for testing eDNA samples to meet the needs for surveillance of the parasite. Moreover, we validated the developed assay in natura by testing samples derived from filtered water and sediments collected inside and outside LMRP's fenced-off area. Our findings for the first time demonstrate the presence of F. magna's eDNA outside the park's internal fenced-off area.

Development of a Novel Internally Controlled HrpB1 Gene-Based Real-Time qPCR Assay for Detection of Burkholderia pseudomallei.

Melioidosis, caused by category B bioterrorism agent Burkholderia pseudomallei, is a seasonal disease of tropical and subtropical regions with a high mortality rate. An early and culture-independent detection ofB. pseudomalleiis required for the appropriate disease management and prevention. The present study is designed to identify novel and unique sequences ofB. pseudomalleiand development of quantitative polymerase chain reaction (qPCR) assay. A novel B. pseudomallei-specific target sequence was identified by in silico analysis for the qPCR assay development. The specificity of the developed assay was assessed using purified DNA of 65 different bacterial cultures, and the sensitivity was estimated using a cloned target gene. Further, a type III secretion protein HrpB1 (HrpB1) gene-based duplex qPCR assay incorporating suitable extraction and amplification control was developed, and its viability was assessed in different clinical and environmental matrices for the detection of B. pseudomallei. In this study, an 80-nucleotide-longB. pseudomallei-specific region within the geneHrpB1was identified by computational analysis. The developedHrpB1-based qPCR assay was highly specific forB. pseudomalleidetection when evaluated with 65 different bacterial cultures. The sensitivity of the qPCR assay with theHrpB1-recombinant plasmid was found to be five copies per qPCR reaction. The assay's detection limit was found to be 5 × 102 CFU/mL for human blood and urine, 5 × 101 CFU/mL in river water, and 2 × 103 CFU/gm in paddy field soil. The results of the study showed the applicability of a novelHrpB1-based qPCR assay for sensitive and specific detection ofB. pseudomalleiin diverse clinical and environmental samples.

Development of a duplex qPCR assay for the detection of coinfection in eels by Japanese eel endothelial cell-infecting virus and Anguillid herpesvirus-1

Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus 1 (AngHV-1) are virulent pathogens in eel aquaculture farms in the Republic of Korea and coexist in the same host. To date, no diagnostic method has been developed that detects them simultaneously. We developed a duplex real-time polymerase chain reaction (duplex qPCR) assay to detect both JEECV and AngHV-1 simultaneously in a single reaction. Two sets of specific primers and TaqMan probes were designed to target the large T-antigen gene of JEECV and the DNA polymerase catalytic subunit gene of AngHV-1. The assay was optimized by changing the primer and probe concentrations and the annealing temperature of the reaction. The duplex qPCR could specifically amplify the specific genes of the two viruses with high efficiency and coefficient of determination. The assay could detect both viruses at a limit of detection of 8 fg/μL. The diagnostic performance of the developed assay was determined by analyzing 82 clinical samples (72 infected and 10 healthy) and comparing the results with those of conventional PCR and SYBR Green-based qPCR; the results correlated well with each other. The duplex qPCR showed 100% and 98.6% diagnostic sensitivity for JEECV and AngHV-1, respectively. The diagnostic specificity of this assay was validated using two potential viral strains, red sea bream iridovirus and Ostreid herpesvirus-1 microvariant, reported in the aquaculture industry. In contrast, the methods already developed including conventional PCR and qPCR cannot detect both JEECV and AngHV-1 at the same time as they should perform in separate reactions. Thus, the duplex qPCR developed herein is a sensitive and specific diagnostic tool for the simultaneous detection of JEECV and AngHV-1 and has subsequent applications in the health management of eels.

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

Bovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. Results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10 −5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for A1 allele in A2 samples at a 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify A1 allele absence in “A2 milk” by screening commercial product on the market. • The LNA duplex qPCR assay developed could be used to verify A1 allele absence in “A2 milk”. • LNA probes reliably discriminated between A1 and A2 alleles. • The relative limits of detection reach up to 2% (15 copies) for A1 allele in A2 samples at a 95% confidence. • Milk is a challenging matrix for DNA isolation due to the presence of PCR inhibitors that leads to low DNA yield.

Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection

Milk proteins determine important milk technological characteristics. Among caseins, Ƙ-casein has been correlated with fat and protein content and cheese yield. Fourteen Ƙ-caseins variants have been described but the alleles A, B and E are the most important ones due to their frequency and/or influence on the technological aptitudes of milk. Therefore, in the present study two different duplex qPCR assays with locked nucleic acid probes (for positions 13104 and 13124 of the Ƙ-casein gene) were developed for the detection of A, B and E variants. Firstly, DNA isolation method from milk somatic cells and hair was optimised. The developed 13124-qPCR assay showed an increased sensitivity reaching up to 6.7 copies DNA copies/reaction at a 95% confidence level with A, B and E alleles reference samples. The 13104-qPCR assay reached up to 6.7 DNA copies/reaction for A allele reference sample and 67 DNA copies/reaction for B and E samples. Intra-assay variation results were below 6%. Applicability was determined using DNA samples from animals with known genotype for Ƙ-casein (AA, AB, BB, BE, AE, EE) and both assays were able to discriminate among the six genotypes with 100% accuracy. Thus, this qPCR method represents a sensitive and rapid option for the detection of Ƙ-casein alleles in both hair and milk samples.

A new duplex qPCR assay for the quantification of honey bee (Apis mellifera) parasites Nosema ceranae and Nosema apis tested with low dose experimental exposure

Nosema ceranae and Nosema apis are microsporidian parasites that cause disease in the European honey bee. Nosema infection is identified as a potential cause of colony loss by beekeepers. Given the importance of Nosema infection in colony mortality and productivity, developing new and improved ways of detecting and quantifying infection loads is vital. We designed and tested a new duplex qPCR assay for the accurate quantification of both N. ceranae and N. apis utilising TaqMan chemistry and the new gBlock® method for the standards. The assay showed good linearity with natural Nosema infection, and a strong correlation with microscopic spore counts. This new assay has high sensitivity and repeatability and was used to investigate Nosema infection in hive surveys and following low dose experimental exposure. In local hives, we found relativity low levels of N. ceranae and very little N. apis across three sites in Blacksburg, VA. A survey of two bee yards in West Virginia showed much higher levels of N. ceranae, but consistent low levels of N. apis. For the experiment, caged bees from two different hives were fed 100 Nosema spores (or a control solution). Exposed bees were collected after two or five days, and infection was quantified using the new assay. Given the low dose, infection levels were not 100%, with some exposed bees remaining N. ceranae free, while others only developed low level infection. This low dose exposure and subsequent infection status (low level infection or infection free) could provide a new understanding of Nosema infection establishment.

Development of duplex qPCR targeting Carnobacterium maltaromaticum and Vagococcus salmoninarum.

In November 2018, Vagococcus salmoninarum was identified as the causative agent of a chronic coldwater streptococcosis epizootic in broodstock brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery in Wisconsin, USA. By February 2019, the epizootic spread to adjacent raceways containing broodstock lake trout (Salvelinus namaycush), whereby fish were found to be coinfected with Carnobacterium maltaromaticum and V.salmoninarum. To differentiate these two pathogens and determine the primary cause of the lake trout morbidity, a quantitative real-time PCR (qPCR) was developed targeting the C.maltaromaticum phenylalanyl-tRNA synthase alpha subunit (pheS) gene. The qPCR was combined with a V.salmoninarum qPCR, creating a duplex qPCR assay that simultaneously quantitates C.maltaromaticum and V.salmoninarum concentrations in individual lake trout tissues, and screens presumptive isolates from hatchery inspections and wild fish from national fish hatchery source waters throughout the Great Lakes basin. Vagococcus salmoninarum and C.maltaromaticum were co-detected in broodstock brook trout from two tribal hatcheries and C.maltaromaticum was present in wild fish in source waters of several national fish hatcheries. This study provides a powerful new tool to differentiate and diagnose two emerging Gram-positive bacterial pathogens.

Simultaneous detection and differentiation of porcine circovirus 3 and 4 using a SYBR Green І-based duplex quantitative PCR assay

Porcine circovirus 4 (PCV4) was a novel circovirus identified from diseased pigs in 2019 in Hunan Province, China, and PCV3 and PCV4 co-infection has been reported. In order to detect and differentiate PCV3 and PCV4 simultaneously, the SYBR Green І-based duplex quantitative PCR (qPCR) assay was established in the present study. The two viruses could be easily distinguished by different Tm values: 86.5°C for PCV3 and 79°C for PCV4, while other porcine pathogens did not shown specific melting peaks. The detection limits of this duplex qPCR assay were 51.7 copies/μL for PCV3 and 67.7 copies/μL for PCV4, and both of the intra-assay and inter-assay of the CV analysis of this assay were less than 2.0 %. Sixty-four clinical samples from 22 different swine farms were screened by the duplex qPCR assay. The results showed that the positive detection rate of PCV3 was 37.5 % (24/64) and PCV4 was 34.38 % (22/64), and PCV3 and PCV4 co-infection rate was 17.19 % (11/64). The detection rate of the duplex qPCR assay was higher than that of the conventional PCR assay. The duplex qPCR was of high sensitivity and specificity, being able to provide technical support for clinical detection, differential diagnosis and control of PCV3 and PCV4.

A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues.

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses.

The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, the performance of three sets of primers and a hydrolysis probe for hEPO were compared. One set showed adequate specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse gDNA. The assay was duplexed with the detection of horse tubulin α 4A (TUBA4A) gene as an endogenous internal control in order to prevent false-negative results due to poor recovery and storage of extracted DNA and/or qPCR experimental variation. For the extraction of hEPO-plasmid, the QIAGEN Gentra Puregene blood kit was shown to recover the majority (62%) of hEPO-plasmid from spiked horse blood cells. The specificity and limit of detection (LOD) of the duplex qPCR assay were determined in accordance with MIQE guidelines. These findings supported the application of this duplex qPCR assay to the detection of hEPO transgene in horse blood cells.

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

BackgroundAsiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols.ResultsOur assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml− 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml− 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays.ConclusionsWe developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis

Powdery mildew and downy mildew are two devastating diseases on cucumber and other cucurbit crops caused by Podosphaera xanthii and Pseudoperonospora cubensis, respectively. Identification and detection of these pathogens from field and plant material could be significant for the selection of resistant varieties and formulation of disease management strategies. In the present study, a duplex qPCR assay developed for simultaneous detection and quantification of both pathogens from different samples. Two sets of species-specific primers developed for the detection of P. xanthii and P. cubensis pathogens by targeting the internal transcribed spacer (ITS) region of the rDNA gene cluster. The specificity of designed primers was also evaluated against the different microbial, plant, soil, and environmental samples. Initially, the individual assays for P. cubensis and P. xanthii were validated using their corresponding species-specific primers, which amplified the prominent and distinctive products of ~ 705 bp and ~ 290 bp size, respectively. SYBR green-based duplex real-time PCR assay was developed to detect and quantify both mildew pathogens from different field samples. The species-specific oligonucleotide primer sets showed high specificity with melt curve peaks at 85.83 °C and 88.05 °C, for P. xanthii and P. cubensis, respectively. The relative quantification and lowest detection limit of qPCR assays using tenfold diluted plasmid (Csp1 and Csd1) DNA were estimated (0.1 pg/µl) through a standard curve. In this study, the species-specific PCR and qPCR assays in both simplex and duplex formats have been validated successfully. These assays could be useful for efficient detection and quantification of mildew pathogens from the cucumber and other cucurbit crops.

Duplex qPCR assay for detection and quantification of Anaplasma phagocytophilum and Rickettsia spp.

Anaplasma phagocytophilum and Rickettsia spp. are vector-borne zoonotic bacteria, which are clinically important especially in immunocompromised patients. There are large gaps in the current knowledge of their geographic distribution and prevalence in both their vectors and hosts. Our aim was to develop reliable and easy detection method for both these pathogens. We made a new hydrolysis probe based duplex Real-Time PCR assay based on previous studies. We optimized the assays and tested them to provide reliable recommended procedures with a sensitivity to a minimum of 10 target DNA copies per sample. The assays were designed to be specific for A. phagocytophilum and in the same reaction detect multiple species of rickettsiae. We designed gBlock quantification standards that provide the option to identify differences in pathogen load among different samples in subsequent studies.

Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/μL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories.

For over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

Development and evaluation of a duplex TaqMan qPCR assay for detection and quantification of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts

BackgroundA question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances.ResultsThe novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq’s) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq’s between 24 and 33.ConclusionsThis duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

Outbreak of macrolide-resistant mycoplasma pneumoniae in a primary school in Beijing, China in 2018

BackgroundOn 7th June, 2018, a primary school in Beijing, China notified Shunyi CDC of an outbreak of acute respiratory disease characterized by fever and cough among students and resulting in nine hospitalization cases during the preceding 2 weeks. We started an investigation to identify the etiologic agent, find additional cases, develop and implement control measures.MethodsWe defined probable cases as students, teachers and other staffs in the school developed fever (T ≥ 37.5 °C) with cough or sore throat; or a diagnosis of pneumonia during May 1–June 31, 2018. Confirmed cases were probable cases with Mycoplasma pneumoniae detected in oropharyngeal (OP) swabs by quantitative real-time polymerase chain reaction (qPCR). We searched case by reviewing school absenteeism records and interviewing students, teachers and staff in this school. Oropharyngeal swabs were collected from symptomatic students. Two qPCR) assay, a duplex qPCR assay, and sequencing were performed to determine the pathogen, genotype and macrolide resistance at the gene level, respectively.ResultsFrom May 1st to June 31st, 2018, we identified 55 cases (36 probable and 19 confirmed), of whom 25 (45%) were hospitalized for complications. All cases were students, none of the teachers and other staffs in the school were with similar symptoms. The attack rate (AR) was 3.9% (55/1398) for all students. The cases were mainly male (58%), with an age range of 7–8 years (median: 7 years). 72% (18/25) of inpatients had radiograph findings consistent with pneumonia, and some cases were hospitalized for up to 4 weeks. Pathogen detection results indicated that Mycoplasma pneumonia (M. pneumoniae) P1 type 1 was the causative agent in this outbreak, and the strain harbored one point mutation of A to G at position 2063.ConclusionsThe infections by macrolide-resistant M. pneumoniae are not always mild and pneumonia was common and M. pneumoniae could causes serious complications which require long-term hospitalization. In the future infectious disease prevention and control practice, M. pneumoniae should be paid more attention. It is necessary to establish and improve the pathogen and drug resistance surveillance system in order to prevent and control such mutated strains of M. pneumoniae from causing future outbreaks or epidemics in China.

Duplex TaqMan Hydrolysis Probe-Based Molecular Assay for Simultaneous Detection and Differentiation of Burkholderia pseudomallei and Leptospira spp. DNA.

Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.

A duplex PCR assay for the simultaneous quantification of Bacteroides HF183 and crAssphage CPQ_056 marker genes in untreated sewage and stormwater

The HF183 marker gene, derived from the 16S rRNA gene of Bacteroides dorei, has been widely used to identify sewage pollution in environmental waters. CrAssphages are recently discovered DNA bacteriophages that are highly abundant in untreated sewage and have shown promises for tracking sewage contamination in environmental waters. In this paper, we report the development of a duplex quantitative PCR (qPCR) assay for simultaneous quantification of HF183 and crAssphage CPQ_056 marker genes in untreated sewage and sewage impacted stormwater. Same primer and probe sequences were used in the duplex qPCR assay as used in published simplex qPCR assays. The performance characteristics of the duplex qPCR assay were similar to its simplex counterparts. We validated the performance of the duplex assay in a collaborative laboratory study with the aim to evaluate reproducibility, sensitivity and concordance for field study. The concordance values between the simplex vs. duplex qPCR assays for HF183 and crAssphage CPQ_056 marker genes ranged from 96.7 to 100% and the mean concentrations of HF183 and CPQ_056 in environmental water samples were remarkably similar or in some cases slightly greater for the duplex qPCR assay suggesting the reliability of this assay for monitoring HF183 and CPQ_056 simultaneously. The newly developed duplex qPCR assay will be a valuable addition to the MST toolbox for sewage pollution monitoring and would allow rapid and comparative sample analysis.