Comparison Of qPCR Research Articles

Comparison of qPCR and chromogenic culture methods for rapid detection of group B streptococcus colonization in Vietnamese pregnant women

IntroductionNeonatal infections can rapidly become severe, with delays in treatment often proving fatal. Streptococcus agalactiae (Group B Streptococcus, GBS) is a common cause, typically transmitted from colonized pregnant women to neonates during childbirth. In Vietnam, routine prenatal care lacks standardized GBS screening protocols. This study aims to compare enhanced qPCR methods with the culture method, evaluate the diagnostic accuracy of these qPCR procedures, and assess the frequency of GBS infection in pregnant Vietnamese women during their final trimester. Materials and methodsPregnant women aged 35 weeks gestation or more were recruited. Rectovaginal swabs were collected and analyzed for GBS using chromogenic culture, a commercial real-time PCR kit, and in-house real-time PCR assays targeting the cfb and sip genes. Clinical diagnostic values were calculated, and GBS prevalence was determined. ResultsThe study included 259 pregnant women with a mean age of 30.2 ± 5.0 years. Of these, 96.6 % had gestational ages of 37 weeks or more at delivery. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the cfb-based and sip-based qPCR assays were 94.1/92.7, 99.0/99.5, 97.1/98.5, 97.8/97.3, and 97.6 %, respectively. The Kappa values were excellent (0.940 and 0.939), with results available in under 2 h. GBS prevalence was 24.7 % and 25.5 % by cfb-based and sip-based qPCR assays, aligning with the culture method (25.5 %). ConclusionsBoth direct real-time PCR assays demonstrated high accuracy and were comparable to chromogenic culture in diagnosing GBS. A significant prevalence of GBS colonization was found among Vietnamese pregnant women in their final trimester.

B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae

Abstract Background In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.). Methods We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA). Results The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1). Conclusions The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.

Comparison of qPCR and metabarcoding for environmental DNA surveillance of a freshwater parasite.

Analysis of environmental DNA (eDNA) has been successfully used across freshwater ecological parasitology to inform management of ecologically and economically important species. However, most studies have used species-specific quantitative polymerase chain reaction (qPCR) assays to detect target taxa. While generally effective, this approach limits the amount of community and management-supporting data that can be obtained from eDNA samples. If eDNA metabarcoding could be conducted with the same accuracy of a single species approach, researchers could simultaneously detect a target species while obtaining vast community data from eDNA samples. We sampled 38 freshwater sites on Fort McCoy, Wisconsin and compared qPCR to metabarcoding for eDNA detection of the ectoparasitic gill louse Salmincola edwardsii, an obligate parasite of Salvelinus fishes (chars). We found no evidence to suggest S. edwardsii occupancy or detection probabilities differed between qPCR and metabarcoding. Further, we found that the number of S. edwardsii reads from metabarcoding were negatively predictive of C T values from qPCR (C T value indicates cycle a significant amount of target eDNA is detected, with lower C Ts indicative of more DNA), demonstrating that our metabarcoding reads positively predicted qPCR DNA quantities. However, the number of reads was not predictive of overall qPCR score (number of positive qPCR replicates). In addition to S. edwardsii, metabarcoding led to the detection of a vast community of over 2600 invertebrate taxa. We underscore the necessity for conducting similar analyses across environments and target species, as the ecology of eDNA will vary on a per-study basis. Our results suggest that eDNA metabarcoding provides a highly sensitive and accurate method for detecting parasitic gill lice while also illuminating the broader biological community and co-occurrence of species in the environment.

Comparison of qPCR and metagenomic sequencing methods for quantifying antibiotic resistance genes in wastewater.

Surveillance methods of circulating antibiotic resistance genes (ARGs) are of utmost importance in order to tackle what has been described as one of the greatest threats to humanity in the 21st century. In order to be effective, these methods have to be accurate, quickly deployable, and scalable. In this study, we compare metagenomic shotgun sequencing (TruSeq DNA sequencing) of wastewater samples with a state-of-the-art PCR-based method (Resistomap HT-qPCR) on four wastewater samples that were taken from hospital, industrial, urban and rural areas. ARGs that confer resistance to 11 antibiotic classes have been identified in these wastewater samples using both methods, with the most abundant observed classes of ARGs conferring resistance to aminoglycoside, multidrug-resistance (MDR), macrolide-lincosamide-streptogramin B (MLSB), tetracycline and beta-lactams. In comparing the methods, we observed a strong correlation of relative abundance of ARGs obtained by the two tested methods for the majority of antibiotic classes. Finally, we investigated the source of discrepancies in the results obtained by the two methods. This analysis revealed that false negatives were more likely to occur in qPCR due to mutated primer target sites, whereas ARGs with incomplete or low coverage were not detected by the sequencing method due to the parameters set in the bioinformatics pipeline. Indeed, despite the good correlation between the methods, each has its advantages and disadvantages which are also discussed here. By using both methods together, a more robust ARG surveillance program can be established. Overall, the work described here can aid wastewater treatment plants that plan on implementing an ARG surveillance program.

Quantification of the percentage proportion of individual animal species in meat products by multiplex qPCR and digital PCR

Determining the composition of food and verifying the manufacturer's declaration continues to represent a methodological challenge. Various methods based on the analysis of proteins (peptides), chemical examination or even DNA detection fail to analyze a complex sample composed of more than one species. Since manufacturers declare the composition of products in percentage weight, the problem for all analytical methods is to determine the 100% muscle and fat tissues containing different amounts of DNA, as water or ice do not contain any DNA. The aim of this study was, therefore, to investigate the possibility of quantifying the percentage of the dominant meat species (pork, chicken and beef) in samples of 2- and 3-component mixtures and in meat products. Two different calculation methods were applied to qPCR and it was shown that a calibration curve is not necessarily required for composition evaluation. Comparison of qPCR and ddPCR showed that these two methods provide comparable data with a low level of discrepancy. The data obtained by qPCR and ddPCR were compared with each other and with the theoretical number of genomic copies. The study showed that the percentage of meat constituents in products with high muscle content can be quantified with high confidence even without taking the heat treatment into account. It was, however, shown that fat can negatively affect the calculation of component proportions and complicate the interpretation of the data.

A comparison of qPCR and microscopy for the detection and enumeration of Cryptosporidium oocysts from drinking water.

Introduction. Cryptosporidium presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of Cryptosporidium is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures.Hypothesis. The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR.Aim. We aimed to develop and evaluate a qPCR for the detection and enumeration of Cryptosporidium in drinking water, and to compare the assay with the standard method used in the UK.Methodology. We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for Cryptosporidium genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 Cryptosporidium oocysts in 10 l of artificially contaminated drinking water.Results. The results demonstrated that detection of Cryptosporidium by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy.Conclusions. Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for Cryptosporidium analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.

WS13.02 Comparison of qPCR and 16S rRNA marker-gene Next-Generation Sequencing (NGS) for the detection and quantification of respiratory pathogens in the sputum of people with cystic fibrosis (PwCF)

Objectives: Non-culture methods are increasingly used for the detection and quantification of pathogens [ 1 Forbes et al. Front Microbiol. 2017; 8: 1069 Crossref PubMed Scopus (177) Google Scholar ]. qPCR allows absolute quantification of target genes, while 16S rRNA marker-gene MiSeq sequencing (NGS) provides taxonomic resolution to a genera level [ 2 Janda JM Abbott SL J Clin Microbiol. 2007; 45: 2761-2764 Crossref PubMed Scopus (1194) Google Scholar ], with the relative abundance of each of these bacterial genera determined within a sample. The aim of this study was to compare qPCR and relative abundance of 16S rRNA amplicons for the detection and quantification of bacteria within sputum samples from PwCF.

Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy.

Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.

Highly sensitive environmental DNA detection of topmouth gudgeon, Pseudorasbora parva: a comparison of qPCR and microfluidic qdPCR

Topmouth gudgeon is a freshwater fish species native to East Asia. Nowadays, P. parva is spread throughout Europe which is of concern because besides being considered one of the worst aquatic Invasive Alien Species (IAS) in Europe it is also a known vector of Spherotecum destruens, the rosette-like parasite lethal to other fish species. The present study describes the development and validation of a new species-specific assay based on hydrolysis probe chemistry to detect P. parva environmental DNA (eDNA) in water samples collected in a northern region of Italy (Friuli Venezia Giulia). Water samples were collected from 55 sites in an area where partial information on the occurrence of the species is available. eDNA was isolated from all samples and the presence of P. parva eDNA was tested by means of qPCR (quantitative PCR) and microfluidic qdPCR (quantitative digital PCR) techniques. Field results for both qPCR and qdPCR were largely in agreement in terms of detection (presence/absence). Thus, we judged the presence/absence by combining the results from the two methods and found that nine sites showed “strong positive” signal of P. parva eDNA (at least 2 positive replicates), 3 showed “suspected” (only 1 positive replicate), and 42 showed “absent”. The current study shows the strong potential of the newly developed eDNA approach to be a valuable addition to the monitoring of the highly invasive topmouth gudgeon in freshwater ecosystems.

Development and comparison of qPCR and qLAMP for rapid detection of the decapod iridescent virus 1 (DIV1)

Decapod iridescent virus 1 (DIV1) is a new virus discovered in recent years that infects farmed shrimp. DIV1 is highly infectious and causes substantial economic loss to the aquaculture industry of China. To prevent and control the spread and outbreak of DIV1 in a timely manner, it is necessary to establish an efficient method for DIV1 diagnosis. In this study, quantitative real-time polymerase chain reaction (qPCR) and quantitative real-time loop-mediated isothermal amplification (qLAMP) detection methods were established based on the specific sequence of the viral ATPase gene. The results indicated that the minimum detection limits of qPCR and qLAMP were 1.9 × 101 copies/μL and 1.9 × 102 copies/μL, respectively; the designed primer had good specificity for DIV1 and did not react with 13 other viruses, including white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreatic necrosis disease (AHPND), infectious hypodermal and haematopoietic necrosis virus (IHHNV), etc. A total of 43 clinical samples suspected of DIV1 infection were diagnosed by qPCR and qLAMP. Our qPCR demonstrated results consistent with a qPCR assay published previously, and the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of qLAMP were 85.71% and 100%, respectively. This result indicates that qPCR and qLAMP have good accuracy in the detection of DIVI in clinical samples. As established in this study, qPCR and qLAMP combined with a comprehensive comparative analysis can provide effective new solutions for the detection of DIV1.

Comparison of qPCR and Metabarcoding Methods as Tools for the Detection of Airborne Inoculum of Forest Fungal Pathogens.

Forest diseases caused by invasive fungal pathogens are becoming more common, sometimes with dramatic consequences to forest ecosystems. The development of early detection systems is necessary for efficient surveillance and to mitigate the impact of invasive pathogens. Windborne spores are an important pathway for introduction of fungal pathogens into new areas; the design of spore trapping devices adapted to forests, capable of collecting different types of spores, and aligned with development of efficient molecular methods for detection of the pathogen, should help forest managers anticipate new disease outbreaks. Two types of Rotorod samplers were evaluated for the collection of airborne inoculum of forest fungal pathogens with a range of spore sizes in five forest types. Detection was by specific quantitative PCR (qPCR) and by high-throughput sequencing (HTS) of amplified internal transcribed spacer sequences using a new bioinformatic pipeline, FungiSearch, developed for diagnostic purposes. Validation of the pipeline was conducted on mock communities of 10 fungal species belonging to different taxa. Although the sensitivity of the new HTS pipeline was lower than the specific qPCR, it was able to detect a wide variety of fungal pathogens. FungiSearch is easy to use, and the reference database is updatable, making the tool suitable for rapid identification of new pathogens. This new approach combining spore trapping and HTS detection is promising as a diagnostic tool for invasive fungal pathogens.

Comparison of qPCR and traditional beach monitoring methods at select Great Lakes beaches and the impact on beach management

Campbell A, Kleinheinz G. 2020. Comparison of qPCR and traditional beach monitoring methods at select Great Lakes beaches and the impact on beach management. Lake Reserv Manage. 36:155–168. The US EPA has adopted numerous techniques for enumerating fecal indicator bacteria, each with advantages and disadvantages. Traditional methods require 18 to 24 h of incubation and do not provide results in time to adequately protect public health. New rapid quantitative polymerase chain reaction (qPCR) methods have emerged. Some initial studies of these rapid methods have shown them to correlate with results from culture-based methods, accurately predicting risk for gastrointestinal illness. However, there are few comparative studies that have been conducted in the Great Lakes at beaches that are not directly impacted by human fecal contributions. In this study, Enterococcus qPCR Method 1609 and Escherichia coli qPCR Method C were compared to the defined substrate methods Enterolert and Colilert, respectively, at 4 beaches on Lake Michigan. Results suggest that no one size fits all for these methods, and each beach behaves very differently depending on the microbiological method chosen. Additionally, we investigated the management impact of changing methods using current regulatory standards. If adopted as the regulatory method, qPCR Method 1609 would result in a minimum increase of beach exceedances (advisories and closures) of 17% and a maximum increase of 43%. Likewise, E. coli Method C has a minimum increase of beach exceedances of 27% and a maximum increase of 47%. This suggests that qPCR may be more protective of public health than current culture-based enumeration methods. The increases in beach closures may have significant ramifications in local communities. This study suggests that new qPCR methods may have a role in replacing current methods, but a site-specific evaluation should be conducted before they are adopted.

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue.Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL.Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels.In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.

Comparison of qPCR and amplicon sequencing based methods for fecal source tracking in a mixed land use estuarine watershed

Fecal indicator bacteria are commonly used to evaluate water quality and make decisions on designating and restricting use.

Minimal Residual Disease Detection at RNA and Leukemic Stem Cell (LSC) Level. Comparison of Qpcr, d-PCR and CD26 Stem Cell Measurements in Chronic Myeloid Leukemia (CML) Patients in Deep Molecular Response (DMR)

Background. A Deep Molecular Response (DMR), defined as BCR-ABL1 p210 transcript at levels <= 0.01% by QPCR, is the prerequisite for a successful interruption of treatment in patients with CML. However, approximately 50% of patients in Treatment Free Remission (TFR) studies had to resume therapy after BCR-ABL1 p210 transcript levels rose above the 0.01% DMR threshold. To improve transcript detection sensitivity, transcript levels were analyzed using D-PCR. D-PCR increased BCR-ABL1 transcript detection sensitivity by 10-100 fold, however its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapse may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally low or silent, insensitivity to tyrosine kinase inhibitors (TKIs) or other CML-causing transcripts not easily detectable by BCR-ABL1 PCR techniques Another approach is to use flow cytometry to detect and quantify bone marrow Ph+ LSCs CD34+/CD38− that co-express CD26 (dipeptidylpeptidase-IV) although its meaning in TFR is unclear.Aim. To compare and examine values of the three different methods of detecting minimal residual disease (MRD) in CML at RNA and LSC levels in patients in TFR or DMR.Patients and Methods. The twenty-seven patients in this study received treatment with either Imatinib (12), Dasatinib (6), Nilotinib (7), Bosutinib (1) or Interferon (1). Twelve patients were in TFR, while the rest were in DMR. TFR patients had stopped therapy for less than 1 year (3), <3 years (2), 6 years (6) and 17 years (1). Blood samples were collected and tested 3 times. BCR-ABL1 transcript quantification was performed using the automated Xpert Ultra BCR-ABL1 MonitorTM Cepheid method and calibrated for the ABL reference gene and BCR-ABL1 target gene. Samples were analyzed according to the manufacurer with results expressed in BCR-ABL1/ABL %IS, or undetectable transcript (U), with PCR sensitivity of 5.0 (>250,000 ABL copy numbers).D-PCR analysis used TaqMan-MGB probes targeting the BCR-ABL1 transcript. A custom assay was designed and produced with a FAM-label, basing on the sequence of routinely-used probes. BCR-ABL1 quantifications were performed analyzing 50ng of cDNA on a QuantStudio 3D Digital PCR System (ThermoFisher Scientific). BCR-ABL1 transcript values using dPCR were expressed as number of copies/ul. The secondary analysis were carried out by AnalysisSuite Cloud Software (ThermoFisher Scientific).To detect peripheral blood circulating CD34+/CD38-/CD26+ LSCs, cells were incubated with anti-CD45 (BD Biosciences), anti-CD34 (581), anti-CD38 (HIT2) and anti-CD26 (M-A261) (BD Pharmigen). Acquisition and analysis were performed by FACSCanto II flow cytometer (BD Biosciences, NR Nannini). CD45+ cells acquired for each sample ranged from 500,000 to 1,000,000. Isotype controls were included in each staining. Median absolute number of CD26+ cells/μL were calculated as follows: (# WBCs/μL) × (% CD34+/CD38−/CD26+ stained CD45+ cells).Results. TFR status, the type of TKI treatment, QPCR results, dPCR data and LSCs quantification are summarized in Table 1.Both d-PCR and LSCs showed higher sensitivity than QPCR, exhibiting positive results when transcript levels using QPCR were undetectable (16). None of the patients tested negative with d-PCR, however 14/16 were under the threshold of 0.468 copies/uL, corresponding to a stable DMR. LSC levels were negative in 5 patients, 3 of which also tested negative with QPCR. In all patients QPCR ranged from 0-0.0068, d-PCR from 0.073 to 0.943, and LSCs from 0 to 0.156.Results were divided in quartiles, depending on molecular response for QPCR, the 0.468 threshold for dPCR and the distribution for the LSCs. The 2 lowest quartiles defined the lowest detectable DMR. A strong correlation of these data in TFR patients was noted (10/12 concordant) while 8/15 DMR patients were discordant.Conclusions To our knowledge this is the first attempt to analyze and compare DMR in a CML population using standard (QPCR) and highly sensitive (dPCR and LSCs) methods. TFR patients, some lasting up to 17 years, were in the lowest detectable DMR categories. Very little is known about the biology of CML implications of circulating LSCs. Larger studies and dynamic scoring will help define their informative and predictive value. [Display omitted] DisclosuresAbruzzese:Pfizer: Consultancy; Ariad: Consultancy; Novartis: Research Funding; BMS: Consultancy.

A comparison of qPCR and ddPCR used for quantification of the JAK2 V617F allele burden in Ph negative MPNs

Philadelphia-negative myeloproliferative neoplasms (MPNs) are a diverse group of diseases whose common feature is the presence of V617F mutation of the JAK2 gene. In the era of novel therapeutic strategies in MPNs, such as JAK-inhibitor therapy, there is a growing need for establishing high sensitive quantitative methods, which can be useful not only at diagnosis but also for monitoring therapeutic outcomes, such as minimal residual disease (MRD). In this study, we compared the qPCR and ddPCR methods and their clinical utility for diagnosis, prognostication, and treatment monitoring of MPNs with JAK2 V617F mutation in 63 MPN patients of which 6 were subjected to ruxolitinib treatment. We show a high conformance between the two methods (correlation coefficient r = 0.998 (p < 0.0001)). Our experiments revealed high analytical sensitivity for both tests, suggesting that they are capable of detecting the JAK2 V617F mutation at diagnosis of MPN with a limit of detection (LoD) of 0.12% for qPCR and 0.01% for ddPCR. The alterations of JAK2 V617F allele burden in patients treated with ruxolitinib were measured by both methods with equal accuracy. The results suggest an advantage of ddPCR in monitoring MRD because of allele burdens below the LoD of qPCR. Overall, the clinical utility of qPCR and ddPCR is very high, and both methods could be recommended for the routine detection of the V617F mutation at diagnosis, though ddPCR will probably supersede qPCR in the future due to cost-effectiveness.

Comparison of qPCR and culture methods for group B Streptococcus colonization detection in pregnant women: evaluation of a new qPCR assay

BackgroundStreptococcus Group B (GBS) colonization in pregnant women is the most important risk factor for newborn disease due to vertical transmission during delivery. GBS colonization during pregnancy has been implicated as a leading cause of perinatal infections. Traditionally, pregnant women are screened for GBS between 35 and 37 weeks of gestation. However, antenatal culture-based screening yields no information on GBS colonization status and offers low predictive value for GBS colonization at delivery. Numerous assays have been evaluated for GBS screening in an attempt to validate a fast and efficient method. The aim of this study was to compare bacteria isolation by culture and two qPCR techniques, targeting sip and cfb genes, respectively, for detecting colonizing GBS.MethodsCultures – the gold-standard technique, a previous qPCR technique targeting the sip gene, and a new proposed qPCR assay targeting the cfb gene were evaluated as diagnostic tools on 320 samples.ResultsConsidering cultures as the gold standard, the evaluated qPCR method detected 75 out of 78 samples, representing a sensitivity of 93.58% (95% confidence interval (CI), 90.89–96.27) and specificity of 94.62% (95% CI, 91.78–97.46). However, an additional analysis was performed for true positives that included not only samples showing positives by culture but samples showing positive for both qPCR assays. The sensitivity and specificity were recalculated including these discrepant samples and a total of 89 samples were considered as positive, giving a prevalence of 27.81%. With this new analysis, the qPCR targeting the cfb gene showed a sensitivity of 95.5% (95% CI, 88.65–98.59) and specificity of 99.13% (95% CI, 96.69–99.97).ConclusionsThe new qPCR method is a sensitive and specific assay for detecting GBS colonization and represents a valuable tool for identifying candidates for intrapartum antibiotic prophylaxis. Cultures should be retained as the reference and the routine technique because of its specificity and cost analysis ratio, but it would be convenient to introduce PCR techniques to check negative culture samples or when an urgent detection is required to reduce risk of infection among infants.

Molecular and serological dynamics of Chlamydia pecorum infection in a longitudinal study of prime lamb production.

BackgroundChlamydia pecorum is a globally significant livestock pathogen causing pathology and production losses. The on-farm infection and serological dynamics and the relevance of existing diagnostic tools for diagnosing C. pecorum in livestock remains poorly characterized. In this study, we characterized the antigen and antibody dynamics of this pathogen in a longitudinal study of prime lamb production, utilizing the infection focused C. pecorum-specific 16S rRNA qPCR assay and serology based chlamydial Complement fixation Test (CFT).MethodsThe study consisted of 76 Border Leicester mixed sex lambs (39 females and 37 males) that were sampled bimonthly from 2–10 months of age in a commercial farm operating in Central NSW, Australia. Blood/plasma was analysed for CFT antibodies, and swabs from conjunctival, rectal and vaginal sites were analysed for C. pecorum shedding using qPCR. We assessed the temporal and overall dynamics of C. pecorum in lambs, including detailed description and comparison of qPCR and CFT, the timing of first detection by either diagnostic method, the lag between infection and antibody response; and the distribution of qPCR load and CFT antibody titre over time.ResultsOver the study period, C. pecorum was highly prevalent (71.0% by qPCR, 92.1% by CFT, 96.0% by both), with 21.1% (16/76) lambs shedding ≥1, 000 qPCR copies/µl (denoted as high shedders). C. pecorum shedding (as evidence of infection) were first observed at two months of age (14.4%) with a significant peak of infection occurring at six months of age (34.2%), whereas seroconversions peaked at eight months of age (81.5%). 52.6% of C. pecorum qPCR and CFT positive lambs became qPCR negative by 10 months of age, indicating clearance of chlamydial infection. Although CFT is utilised for on-farm detection of active infection, we confirm that it lagged behind qPCR detection (average lag 1.7 ± 2.1 months) and that the proportion of qPCR positives simultaneously identified by CFT was low with 2/11 (18.1%), 0/13, 17/25 (68.0%), 5/7 (71.4%) and 1/10 (10.0%) concurrent seroconversions occurring at two, four, six, eight and 10 months of age, respectively.DiscussionThis work reveals rapid rates of C. pecorum infection and widespread exposure during lamb production. The comparison of molecular and serological diagnostic agreement longitudinally, supports the use of qPCR as an important ancillary tool for the detection of active infections in conjunction with chlamydial CFT for routine veterinary diagnostics. Development of rapid Point-of-Care (POC) tools for diagnosing active infection would be valuable for producers and veterinarians.

Development of a time-effective and highly specific quantitative real-time polymerase chain reaction assay for the identification of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in artisanal raw cow’s milk cheese

The first objective of this work included the development of real-time polymerase chain reaction (RT-PCR) which is also known as quantitative polymerase chain reaction (qPCR) assays to quantify two species of lactic acid bacteria which play a very important role in cheese ripening: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The second objective was the comparison of qPCR and plate counts of these two species present in raw cow’s milk cheese samples during different stages of ripening. Thirty-three deoxyribonucleic acid (DNA) samples coming from seven different bacterial species, which were phylogenetically related or commonly isolated from raw milk and dairy products, were chosen as positive and negative controls. The qPCR assays showed a high quantification capacity characterised by their linearity (R2 &gt; 0.998), PCR efficiencies which were within the range 78.0–90.0% for L. delbrueckii subsp. bulgaricus, and 93.6–100.5% for S. thermophilus, and quantification limit (103 gene copies/ml for L. delbrueckii subsp. bulgaricus and 10 gene copies/ml for S. thermophilus). The importance of our study is in the monitoring of changes in populations of L. delbrueckii subsp. bulgaricus and S. thermophilus contributing to cheese ripening using the newly designed qPCR assay.

Sources of variability in the measurement of Ascaris lumbricoides infection intensity by Kato-Katz and qPCR

BackgroundUnderstanding and quantifying the sources and implications of error in the measurement of helminth egg intensity using Kato-Katz (KK) and the newly emerging “gold standard” quantitative polymerase chain reaction (qPCR) technique is necessary for the appropriate design of epidemiological studies, including impact assessments for deworming programs.MethodsRepeated measurements of Ascaris lumbricoides infection intensity were made from samples collected in western Kenya using the qPCR and KK techniques. These data were combined with data on post-treatment worm expulsions. Random effects regression models were used to quantify the variability associated with different technical and biological factors for qPCR and KK diagnosis. The relative precision of these methods was compared, as was the precision of multiple qPCR replicates.ResultsFor both KK and qPCR, intensity measurements were largely determined by the identity of the stool donor. Stool donor explained 92.4% of variability in qPCR measurements and 54.5% of observed measurement variance for KK. An additional 39.1% of variance in KK measurements was attributable to having expelled adult A. lumbricoides worms following anthelmintic treatment. For qPCR, the remaining 7.6% of variability was explained by the efficiency of the DNA extraction (2.4%), plate-to-plate variability (0.2%) and other residual factors (5%). Differences in replicate measurements by qPCR were comparatively small. In addition to KK variability based on stool donor infection levels, the slide reader was highly statistically significant, although it only explained 1.4% of the total variation. In a comparison of qPCR and KK variance to mean ratios under ideal conditions, the coefficient of variation was on average 3.6 times larger for KK highlighting increased precision of qPCR.ConclusionsPerson-to-person differences explain the majority of variability in egg intensity measurements by qPCR and KK, with very little additional variability explained by the technical factors associated with the practical implementation of these techniques. qPCR provides approximately 3.6 times more precision in estimating A. lumbricoides egg intensity than KK, and could potentially be made more cost-effective by testing each sample only once without diminishing the power of a study to assess population-level intensity and prevalence.