Quantitative PCR Assay For Detection Research Articles

Development and Evaluation of Real-Time Quantitative PCR Assays for Detection of Phellinus noxius Causing Brown Root Rot Disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non-Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

Development and validation of a quantitative PCR assay for detection of Sulawesi tortoise adenovirus

In 2007, a mortality event involving over 100 Sulawesi tortoises (Indotestudo forsteni), two Impressed tortoises (Manouria impress) and a critically endangered Burmese star tortoise (Geochelone platynota) was attributed to Sulawesi tortoise adenovirus (STADV; genus Siadenovirus). We developed a TaqMan quantitative PCR assay targeting the DNA polymerase gene of STADV for use in clinical diagnosis and epidemiologic surveillance. This assay failed to amplify five closely-related chelonian adenoviruses, indicating high analytical specificity. The assay performed with high efficiency (slope = −3.337; R2 = 0.999) and high inter- and intra-assay repeatability (coefficient of variation <1.36 % at all standard curve dilutions). Dynamic range included 1.00 × 107 to 1.00 × 101 target copies per reaction and limit of detection was 101 target copies per reaction, though 100 target copies per reaction were intermittently detected. This qPCR assay provides a valuable diagnostic tool for characterization of STADV epidemiology, including potential identification of the North American reservoir host.

Development of real-time PCR assay for quantitative detection of Clostridium septicum

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.

Development of an ultrafast PCR to detect clinically relevant acquired vancomycin-resistance genes from cultured enterococci.

VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA, vanB, vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE.

Multiplex Quantitative PCR Assay for Detection of Spinach and Lettuce Downy Mildews Using Spore Trapping

Downy mildews are major constraints on spinach and lettuce production globally. Disease management can be achieved with fungicides, but routine applications are costly and can lead to pathogen resistance. Detection of airborne spores could guide sustainable fungicide application, and, considering that spinach and lettuce are often grown in the same cycles, simultaneous detection of both pathogens is practical. Here, a multiplex hydrolysis probe quantitative PCR assay was designed using single copy mitochondrial DNA targets for spinach downy mildew ( Peronospora effusa) and lettuce downy mildew ( Bremia lactucae). To quantify P. effusa and B. lactucae sporangia, a standard curve was developed for each pathogen using the multiplex qPCR to amplify DNA obtained from known dilutions of sporangia. Analysis of these curves revealed a greater sensitivity for B. lactucae, indicating that the sporangia of B. lactucae may harbor more mitochondria than those of P. effusa, providing insight into the biology of these pathogens. The multiplex qPCR assay was partnered with two different spore trap types: a cyclone spore trap and an impaction spore trap. Results from air sampling revealed that the cyclone spore traps collect significantly more sporangia compared with impaction traps. Exposure of P. effusa sporangia to desiccation was performed to assess the environmental impact on the assay, and although detection levels were reduced, they were still apparent. This detection and quantification tool will be useful in efforts to improve the accuracy of downy mildew forecasting, which in return may reduce fungicide usage or improve its efficiency. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Comparison of quantitative PCR and digital PCR assays for quantitative detection of infectious bronchitis virus (IBV) genome

The quantitative polymerase chain reaction (qPCR) technique is an extensively used molecular tool for the detection and quantification of viral genome load. However, since the qPCR assay is a relative quantification method that relies on an external calibration curve it has a lower assay precision and sensitivity. The digital PCR (dPCR) technique is a good alternative to the qPCR assay as it offers highly precise and direct quantification of viral genome load in samples. In this study, performance characteristics such as the quantification range, sensitivity, precision, and specificity of the dPCR technique was compared to qPCR technique for the detection and quantification of IBV genome loads in serial dilutions of IBV positive plasmid DNA, and IBV infected chicken tissue and swab samples. The quantification range of the qPCR assay was wider than that of the dPCR assay, however dPCR had a higher sensitivity compared to qPCR. The precision of quantification of DNA in plasmid samples in terms of repeatability and reproducibility of results was higher when using the dPCR assay compared to qPCR assay. The quantification results of IBV genome load in infected samples by the qPCR and dPCR assays displayed a high correlation. Hence, our findings suggest that dPCR could be used in avian virology research for improved precision and sensitivity in detection and quantification of viral genome loads.

Development of TaqMan quantitative PCR assay for detection of Nocardia seriolae in fish and the environment

Nocardia seriolae is a gram-positive bacterium that causes nocardiosis, threatening fish farming. Advanced nocardiosis is challenging to control; thus, accurate detection methods of the causal agent in the early disease stage are required. In this study, we developed a TaqMan fluorescence quantitative PCR (qPCR) assay for quantitative detection of N. seriolae in fish tissues and water samples. A pair of highly specific primers and a TaqMan probe were designed based on the N. seriolae 16S23S rRNA internal transcribed spacer (ITS) region. A high correlation coefficient (R2 = 0.998) of the standard curve with a 99.5% efficiency was obtained. The qPCR detection limit of the method was as low as 19.8 copies/μL, 1000 times more sensitive than conventional PCR, and has a good performance in the detection of cultured bacteria (y = −3.750× + 48.075, R2 = 0.974). Even 1.42 CFU/mL N. seriolae collected from 500 mL of natural pond water can be detected. Furthermore, a linear model for the relationship between the log of bacteria load and Cq values in water was established (y = −3.239× + 40.978), and the R2 value was 0.979. This assay was used for accurate N. seriolae detection in fish tissues, water samples, feeds and soils. This study provides a valuable tool for the early detection and control of nocardiosis in aquaculture.

Development of a real-time quantitative PCR assay for detection and quantification of the marine bacterium Alteromonas macleodii from coastal environments

Alteromonas macleodii is a ubiquitous marine bacterial species found in a variety of habitats that displays both planktonic and particle-associated lifestyles. Transcriptomic studies demonstrate that, even when present at low abundance, it can make significant contributions to biogeochemical cycles, and its specific association with key marine phytoplankton species indicates other ecological roles as well. It has also been shown to be one of the early colonizers of copper-treated marine vessels. There currently exist no rapid, reliable molecular assays for the detection and quantification of A. macleodii from its different environments. We developed a real-time PCR assay, specific to A. macleodii. This assay targets the DNA gyrase B subunit (gyrB) gene, which occurs as a single copy in the genome. The assay possesses an amplification efficiency of 94.3%, with a limit of detection of 2.5 gyrB copies per μL. Assay specificity was validated by melt curve analysis, followed by sequencing of the amplified product. The assay was specific to thirteen A. macleodii strains and did not amplify other marine bacteria, including Roseobacter denitrificans, Silicibacter sp. TM1040, Vibrio coralliilyticus, Vibrio harveyi, and Vibrio alginolyticus. It also did not amplify Alteromonas mediterranea, a close relative that can occur in the same environment as A. macleodii. This assay was used to determine the presence and abundance of A. macleodii from a range of coastal habitats. The assay was also used to monitor the A. macleodii growth in biofilm and planktonic cultures over time in the presence of elevated copper. This assay provides a rapid and reliable means to assess the presence and abundance of a ubiquitous marine bacterium that, even at low abundance, has been shown to make significant contributions to key marine processes.

Development of a rapid and sensitive reverse transcription real-time quantitative PCR assay for detection and quantification of grass carp reovirus II

Hemorrhagic disease of grass carp, which is induced by grass carp reovirus II (GCRV-II), leads to mass mortality in grass carp culture and causes enormous economic loss. However, there is currently no quantitative analysis method for the detection of GCRV-II, which is greatly restricted the etiological and epidemiological study of the disease. In this study a reverse transcription TaqMan PCR (RT-qPCR) assay was developed for the quantitative detection of GCRV-II. The probe and primers targeted location is the segment 6 (S6) region of the GCRV-II genome which is highly conserved. Standard curves were drawn and criteria were confirmed after the determination of the optimum reaction conditions. The species-specific assay showed that the method is highly specific and has no cross reactions with other pathogens. The assay was sufficiently sensitive to detect as low as 10 copies of virus RNA. Moreover, the method has a very good repeatability for batches and inter-batches sample detection. Then the method was applied to detect the virus in tissue samples from clinically infected grass carp, compared with conventional RT-seminested PCR, the RT-qPCR represents a specific value for detection rate of positive samples. In summary, the RT-qPCR was applied and achieved high sensitivity and specificity for GCRV-II detection.

A nested quantitative PCR assay for detection of the hard clam pathogen Mucochytrium quahogii (=QPX) in environmental samples

Progress in understanding and managing QPX disease outbreaks in hard clams (Mercenaria mercenaria) has been limited by lack of insight into basic aspects of the biology and ecology of the opportunistic pathogen Mucochytrium quahogii (formerly QPX or Quahog Parasite Unknown). One barrier is that while several methods have been able to detect M. quahogii in seawater and sediment, its abundance was typically too low for reliable quantification by those methods. Here we describe the development and validation of a sensitive, M. quahogii-specific, nested quantitative PCR (nqPCR) assay following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. The assay reaches the theoretical limit of detection (LOD) of a PCR assay at 3 copies per reaction with excellent efficiency, linearity, and minimal sample PCR inhibition. The functionality of the assay was evaluated by quantifying M. quahogii in sediment and seawater samples, which revealed that M. quahogii was broadly distributed throughout the marine environment, detected in 75% of samples, with mean estimated abundance of 0.21 cells per mg sediment, 0.55 cells per ml bottom seawater, and 0.02 cells per ml surface seawater. M. quahogii was most prevalent and most abundant in sediment and bottom seawater samples, suggesting that the flocculent layer at the sediment-water interface is an important environmental reservoir where M. quahogii may interact with hard clams. This assay will serve as a valuable tool to better understand QPX disease dynamics and offers a model to guide development of similar assays for other important marine microbes typically present at similarly low abundance.

Optimization of Real-Time Quantitative PCR assay for detection of Echinococcus granulosus in fecal samples

Optimization of Real-Time Quantitative PCR assay for detection of Echinococcus granulosus in fecal samples

Abstract 5156: Multiplexed EGFR mutation detection with C797S and T790M allelic context using six-color digital PCR

Abstract Non-small cell lung cancer (NSCLC) patients harboring activating mutations in epidermal growth factor receptor (EGFR) are benefited from Tyrosine Kinase Inhibitor (TKI) targeted therapy. However, more than 50% of patients receiving the first or second generations of TKI develop a resistance mutation, EGFR-T790M, and they will need to switch to the third-generation TKI (Osimertinib). Upon treatment, a second acquired mutation, EGFR-C797S, leads to the resistance to Osimertinib. If the C797S and T790M mutations are located in trans, a combination of the first and third-generation of TKI(s) may offer some benefit to the patient. The impact of the allelic context to the subsequent treatment is yet to be established and thus an assay identifying C797S and T790M allelic context may serve as a useful research tool. In recent years, digital PCR has emerged to be an ultra-sensitive method in non-invasive identification and monitoring of cancer mutations. However, compared to qPCR and NGS methods, current digital PCR assays are limited by the number of biomarkers that can be incorporated in a single assay and the maximum sample volume that can be screened in one test. In this study, we describe a multiplex digital PCR assay for quantitative detection of EGFR sensitizing and resistance mutations, and identification of C797S and T790M allelic context, using a 6-color digital PCR system that allows high sample volume utilization. The sensitivity and specificity of this multiplex dPCR assay were tested using a panel of contrived cell-free DNA samples. Individual mutations can be detected at as low as 0.2% fractional abundance level. Furthermore, we compared the performance of the multiplex with the 2-color droplet digital PCR Expert Design Assays on the Bio-Rad QX200 ddPCR system, using a commercial multiplex cfDNA reference standard set. The multiplexed EGFR mutation assay on the 6-color Digital LightCycler® System is a promising method for rapid and sensitive monitoring of EGFR mutations in cell-free DNA. Citation Format: Wei Yang, Mari Christensen, Jennifer K. Chan, Julie Tsai, Nancy Patten, Ha B. Tran, Grant R. Hillman, Yu Chuan Tai, Patrick Bogard, Claudia M. Litterst, Victoria H. Brophy, Nick Newton, Christopher D. Nelson, Wouter J. Pattje. Multiplexed EGFR mutation detection with C797S and T790M allelic context using six-color digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5156.

Investigation of human parvovirus B19 prevalence in a large healthy umbilical cord blood donors.

Background and Objectives:Umbilical cord blood (UCB) was used to source hematopoietic stem cells in the past. Despite the apparent advantages of UCB transplantation, virus reactivation poses a considerable danger in allogeneic hematopoietic stem cell transplantation (HSCT). Human Parvovirus B19 is regarded as a potential threat to UCB contamination. This study aimed to evaluate the prevalence of parvovirus B19 in cord blood donors by Semi-Nested PCR. This study is the first largescale report of the B19 DNA in cord blood donors in Iran.Materials and Methods:A total of 691 umbilical cord blood were collected under standard procedure. Then, DNA from buffy coat and plasma were extracted, and semi-nested PCR was performed for all samples.Results:Two out of 691 samples (0.29%) indicated viremia in plasma and buffy coat.Conclusion:In this line, designing and validating a quantitative PCR assay for detection, quantification, and discrimination of Human B19 DNA genotypes of cord blood donors is necessary to enhance the safety of this source of stem cells.

Development and Validation of a Gamma Globin RT-Ddpcr Exploratory Biomarker for Sickle Cell Phase 1 Clinical Trial Fis 002-2020

FTX-6058, a selective and potent binder of embryonic ectoderm development protein (EED), is being investigated in Sickle Cell Disease (SCD). In-vitro and pre-clinical Townes mouse studies show FTX-6058 upregulates expression of the gamma globulin gene leading to increased levels of fetal hemoglobin (HbF). In human clinical trials, as an early indicator of FTX-6058 induced gamma globulin expression, a reverse transcriptase droplet digital PCR (RT-ddPCR) assay was developed and validated by Precision for Medicine. Versus RT-qPCR, RT-ddPCR gives absolute copy number, does not require a standard curve, and is more precise and reproducible for low expressed targets 1,2. Precision for Medicine developed and validated RT-ddPCR assays for the target globin genes α, β, and γ and for the housekeeping genes TFRC and OAZ1. RNA isolated from 3 healthy and 3 SCD affected individuals was used for development and validation of the assay. The upper and lower limits of quantification were 120,000 and 10.5 copies/20μL reaction, respectively. For samples with >50 copies/20μL reaction, the CV for technical replicates was <20% for all transcripts. Furthermore, Precision for Medicine demonstrated SCD affected individuals express approximately 15-fold more γ globulin versus non affected individuals. Fulcrum utilized the globin RT-ddPCR assay validated by Precision for Medicine in the FTX-6058 phase 1 clinical trial FIS 002-2020.1 Zhao Y, Xia Q, Yin Y, Wang Z (2016), Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri. PLoS ONE 11(7): e0159004. doi:10.1371/journal.pone.01590042 Taylor SC, Laperriere G, Germain H (2017), Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports 7(2409): DOI:10.1038/s41598-017-02217-x DisclosuresRoth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Yang: Fulcrum Therapeutics, Inc.: Consultancy. Tsavachidou: Fulcrum Therapeutics, Inc.: Consultancy. Davis: Fulcrum Therapeutics, Inc.: Consultancy.

Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy.

Aims and ObjectivesMore than half of the world’s population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycin-resistant H. pylori as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin- and levofloxacin-resistant H. pylori strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in H. pylori directly from the gastric biopsies.Materials and MethodsSpecific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay’s sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies.ResultsIn this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and −LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively.ConclusionThe effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin- and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.

Development of a dual-labeled, hydrolysis probe-based, real-time quantitative PCR assay for detection of both genotypes of duck circovirus-1 (DuCV-1) and DuCV-2.

In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) assay based on a dual-labeled hydrolysis probe to simultaneously detect both duck circovirus (DuCV) 1 and DuCV-2. The reproducibility, sensitivity and specificity of the primer set and probe were evaluated using other duck pathogens. The detection limit was 20 copies per µL. The intra-assay coefficients of variation (CVs) were ≤ 0.73% and the inter-assay CVs were ≤ 1.89%. No cross-reaction occurred with other duck pathogens. In addition, the qPCR assay was successfully applied to the simultaneous detection of DuCV-1 and DuCV-2 in clinical field samples. Therefore, this assay will be useful for laboratory diagnosis and epidemiological field studies of DuCV.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11262-021-01862-9.

Development and validation of a quantitative PCR assay for detection of Terrapene herpesvirus 2 in eastern box turtles (Terrapene carolina carolina)

Herpesviruses are associated with disease in many chelonian species, resulting in pathology such as respiratory tract infection, stomatitis, conjunctivitis, hepatitis, and papillomatosis. Herpesvirus-associated fibropapillomas cause significant morbidity and mortality in marine turtles, and have been identified in an eastern box turtle (Terrapene carolina carolina) infected with Terrapene herpesvirus 2 (TerHV2). Further investigation is necessary to understand the impact of carcinogenic herpesviruses on chelonian health; however, reliable and specific methods for detection and quantitation of herpesviral load are lacking. The purpose of this study was to develop and validate a quantitative PCR assay for detection of TerHV2 in box turtles. TaqMan primer-probes were developed targeting the DNA polymerase gene. Inter- and intra-assay variability, linear range of detection, limit of detection, and specificity were assessed. The assay was highly specific for TerHV2, failing to amplify seven closely-related chelonian herpesviruses. It performed with high efficiency (slope = -3.52, R2 = 1, efficiency = 92.29 %), low intra-assay variability and low inter-assay variability (coefficient of variation ≤ 1.25 % at all standard dilutions). Reaction efficiency was not impacted in the presence of box turtle DNA from combined oral/cloacal swabs or whole blood. This qPCR assay has a linear range of detection from 107 to 101 viral copies per reaction and provides a valuable tool in the surveillance and characterization of TerHV2 in box turtles.

Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis.

Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi

Lates calcarifer herpes virus (LCHV) is a novel virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per μl of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 4 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry.

Correction to: Development of two quantitative PCR assays for detection of several Cottus species from environmental DNA in Pacifc coast watersheds of North America

In the original publication of the article, the latitude and longitude values of the eDNA sample site, “Thornton Creek, WA” were published incorrectly in Table 2. The corrected Table 2 is given below.