Real-time PCR Assay For Detection Research Articles

A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria

The transfer of biocide and antibiotic resistance genes by mobile genetic elements is the most common mechanism for rapidly acquiring and spreading resistance among bacteria. The qacEΔ1 gene confers the resistance to quaternary ammonium compounds (QACs). It has also been considered a genetic marker for the presence of class 1 integrons associated with multidrug-resistant (MDR) phenotypes in Gram-negative bacteria. In this study, a TaqMan real-time PCR assay was developed to detect the qacEΔ1 gene in Gram-negative bacteria. The assay has a detection limit of 80 copies of the qacEΔ1 gene per reaction. No false-positive or false-negative results have been observed. Simultaneous amplification and detection of the 16S rRNA gene is performed as an endogenous internal amplification control (IAC). The TaqMan real-time PCR assay developed is a rapid, sensitive, and specific method that could be used to monitor resistance to QACs, the spread of class 1 integrons, and the prediction of associated MDR phenotypes in Gram-negative bacteria.

A-302 Evaluation of real-time PCR assays for detection of sexually-transmitted infections

Abstract Background Sexually transmitted infections (STIs) have serious negative consequences for reproductive health worldwide. They are associated with infertility, premature birth and neonatal infections. Five STI pathogens, namely Trichomonas vaginalis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were selected for the evaluation of three potential real-time PCR assays that can be adopted for diagnostic testing. Methods A total of 36 samples were included, consisting of genital swabs, urine, abscesses and samples provided by quality assurance programmes. DNA extraction was performed using an automated nucleic acid extraction system, abGenixTM. Commercial genomic controls and DNA extracts donated by external laboratories were used as well. The extracted nucleic acids were tested using three different assays, namely Seegene Anyplex II STI-5 Detection, Thermo Fisher Scientific customized STI Panel assay, and TIB MOLBIOL STI LightMix Modular kits, according to the manufacturers’ instructions. Results A total of 67% and 11% of the samples showed equivalent positive and negative results, respectively. However, 22% of the samples had non-concordant results. TIB MOLBIOL STI LightMix Modular kit was unable to differentiate between Ureaplasma parvum and Ureaplasma urealyticum in 9 samples. Genomic controls were tested, and Seegene Anyplex II STI-5 Detection was unable to detect lower concentrations of DNA for Trichomonas vaginalis, Mycoplasma genitalium and Mycoplasma hominis. All assays were able to detect lower concentrations of Ureaplasma parvum DNA, but detectable concentrations were higher for Ureaplasma urealyticum. Conclusions In conclusion, the performance of each assay differed according to pathogen. None of the assays offered equal performance for all pathogens. We have adopted Thermo Fisher Scientific customized STI Panel assay in our diagnostic testing due to its advantage of being user friendly and cost effective.

Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale.

The parasite species formerly known as Plasmodium ovale, Plasmodium ovalecurtisi (P. ovalecurtisi) and Plasmodium ovalewallikeri (P. ovalewallikeri), are endemic across multiple African countries. These species are thought to differ in clinical symptomatology and latency, but only a small number of existing diagnostic assays can detect and distinguish them. In this study, we sought to develop new assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri by leveraging recently published whole-genome sequences for both species. Repetitive sequence motifs were identified in available P. ovalecurtisi and P. ovalewallikeri genomes and used for assay development and validation. We evaluated the analytical sensitivity of the best-performing singleplex and duplex assays using synthetic plasmids. We then evaluated the specificity of the duplex assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), and validated its performance using 55 P. ovale samples and 40 non-ovale Plasmodium samples from the DRC. The best-performing P. ovalecurtisi and P. ovalewallikeri targets had 9 and 8 copies within the reference genomes, respectively. The P. ovalecurtisi assay had high sensitivity with a 95% confidence lower limit of detection (LOD) of 3.6 parasite genome equivalents/μl, while the P. ovalewallikeri assay had a 95% confidence LOD of 25.9 parasite genome equivalents/μl. A duplex assay targeting both species had 100% specificity and 95% confidence LOD of 4.2 and 41.2 parasite genome equivalents/μl for P. ovalecurtisi and P. ovalewallikeri, respectively. We identified promising multi-copy targets for molecular detection and differentiation of P. ovalecurtisi and P. ovalewallikeri and used them to develop real-time PCR assays. The best performing P. ovalecurtisi assay performed well in singleplex and duplex formats, while the P. ovalewallikeri assay did not reliably detect low-density infections in either format. These assays have potential use for high-throughput identification of P. ovalecurtisi, or for identification of higher density P. ovalecurtisi or P. ovalewallikeri infections that are amenable to downstream next-generation sequencing.

Varying amplification efficiency real-time PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse

The most common foodborne infection in the world that leads to hospitalizations is caused by Salmonella enterica. Most Salmonella testing workflows are qualitative and only determine the presence or absence of Salmonella, failing to quantify the contamination load present. This study aimed to standardize and validate a novel varying amplification efficiency real-time PCR assay for the detection and estimation of broad Salmonella contamination levels. The approach relies on the use of two Salmonella-specific primer-probe pairs (i.e., invA and ttrR) with different amplification efficiencies to detect and differentiate between samples contaminated with low (1 log CFU/30 mL) or high (2–4 log CFU/30 mL) levels of Salmonella. The standardized multiplex PCR assay was validated with 131 pure culture strains and 260 laboratory-inoculated chicken-rinse samples. The multiplex assay specifically identified all Salmonella strains. The invA and ttrR primer-probe showed amplification efficiency of 128% and 90%, resulting in an analytic sensitivity of 0.01 and 0.1 pg/reaction for pure culture DNA samples, respectively. After a 5-h enrichment, the assay detected Salmonella in all inoculated samples and accurately identified high and low contamination levels in 87% (n = 226/260) of samples. This varying amplification efficiency (VAE) assay offers a simple approach enabling the food industry to detect and identify high-risk samples contaminated by Salmonella.

Real-time PCR primers and probes for the detection of Shiga toxin genes, including novel subtypes

Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes.A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence.The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.

Development and analytical characteristics of a quantitative real-time PCR assay for detection of spheniscid alphaherpesvirus 1 in penguins.

Herpesviruses are associated with disease in many penguin species. Herpesvirus-associated lesions can cause significant morbidity and mortality in penguins and have been identified in African penguins (Spheniscus demersus), Humboldt penguins (Spheniscus humboldti), and a little blue penguin (Eudyptula minor) infected with spheniscid alphaherpesvirus 1 (SpAHV1). Further investigation is necessary to understand the impact of herpesviruses on penguin health, but there are no rapid, sensitive, and specific methods for detecting and quantifying herpesviral load. We therefore developed a quantitative real-time PCR (qPCR) assay for the detection of SpAHV1 in penguins. TaqMan primer-probes targeting the DNA polymerase gene were designed using a commercial software program. Inter- and intra-assay variability, dynamic range, limit of detection, and analytical specificity were assessed. We used our assay to analyze previously collected field samples from Punta San Juan, Peru, in which conventional consensus PCR had detected one SpAHV1-positive penguin sample. Our qPCR assay was highly specific for SpAHV1. It had a dynamic range of 107-101 target copies per reaction and performed with high efficiency and low intra- and inter-assay variability. Reaction efficiency was not impacted by penguin DNA from SpAHV1-negative tracheal swabs. We detected an additional field sample as positive with our newly developed qPCR assay, and although this likely represents detection of another infected penguin, the true disease status of this population is currently uncharacterized given that no gold-standard test exists for SpAHV1. Our qPCR assay may provide a valuable tool in the surveillance and characterization of SpAHV1 in penguins.

Bacteriological evaluation and advanced SYBR-green multiplex real-time PCR assay for detection of minced meat adulteration.

Minced meat is a valuable source of nutrients, but it is vulnerable to contamination by microorganisms commonly present in the environment. In addition, there is a risk of adulteration with cheaper meat sources, which can be harmful to consumers. It is crucial to identify meat adulteration with distinct microbiological analysis for legal, economic, religious, and public health purposes. A total of 100 minced meat samples were collected from several markets in Sharkia Governorate, Egypt. These samples were then subjected to bacteriological testing and an advanced multiplex PCR method. This method enables the detection of bovine, equine, porcine, and dog species in meat samples with just one step. The adulterated samples had a higher total bacterial count and pH values compared to pure bovine meat. These differences in bacterial count and pH values were statistically significant, with p-values of 0.843 (log10) and 0.233, respectively. The frequency of Escherichia coli occurrence was 13%, and the O111 serotype was predominant in the adulterated samples. Listeria monocytogenes and Staphylococcus aureus were isolated with prevalence rates of 3% and 29%, respectively. Besides, the SYBR-green multiplex real-time PCR assay used in this study detected adulteration with dog, equine, and porcine meats in the examined samples at rates of 9%, 5%, and 4%, respectively. This method provides a sensitive and specific approach to detect issues related to well-being and safety.

589. Clinical Utility and Patient Characteristics Associated with Positive Whole Blood PCR for Borrelia spp. in an Endemic Area Using Novel High-Definition PCRTM assay: A Case Series

Abstract Background Current guidelines recommend antibody testing to detect acute borreliosis. Detectable antibody response takes about 2-3 weeks limiting its usefulness in early infection. The ChromaCode High-Definition PCR (HDPCR)™ Tick-Borne Pathogen Panel (TBP) is a research use only qualitative, real-time PCR assay for detection and identification of tick-borne pathogens including Borrelia spp. from whole blood samples. Methods HDPCR™ TBP was put in clinical use after this was validated in the laboratory to identify Anaplasma phagocytophilum, Babesia microti, Ehrlichia spp, Borrelia spp. (B. burgdoferi B. mayonii, and B. miyamotoi). Chart review was performed on all (+)Borrelia spp. from 8/1/22 to 12/31/22. Descriptive analysis was performed on each case. All data was captured and stored using RedCap. Results Over a period of 5 months, 9 (0.5%) out of 1667 tested were (+) for B. burgdorferi; one (0.06%) was (+) for B. miyamotoi; out of these 6 cases had clinical information for review as described in Table 1. Mean age was 64 years; 4 were white and 2 were Hispanic. All 6 lived in western Massachusetts, and only 1 had a known history of tick bite. All had reported systemic symptoms at the time of testing. 3 had (+) B. burgdorferi IgM antibodies;1 case tested (+) PCR for B. miyamotoi but serology was not performed. 1 case was (+) for B. burgdorferi PCR 28 days after symptom onset and had babesiosis co-infection; 1 with B. burgdorferi mono-infection had a (+) PCR test on day 14 of symptoms. All 6 had symptom improvement after doxycycline treatment for 10-14 days. Case description Conclusion HDPCR™ TBP for Borrelia spp is a low yield test, even in an endemic region. All (+) cases were symptomatic and clinically improved following doxycycline treatment supporting the test’s validity. Two cases had negative IgM antibody testing and diagnosis would be likely missed without PCR testing. None were immunocompromised including 2 with prolonged PCR positivity with 1 case having babesiosis co-infection. Study was limited only to 6 cases excluding analysis of other tick-borne infections as part of syndromic testing. Clinical usefulness and timing of HDPCR™ TBP to diagnose borreliosis and other tickborne illnesses need further study. Disclosures All Authors: No reported disclosures

Development and validation of a multiplex real-time PCR assay for detection and quantification of Streptococcus pneumoniae in pediatric respiratory samples

ABSTRACT Streptococcus pneumoniae ( Spn ) is a bacterial pathogen that causes a range of disease manifestations in children, from acute otitis media to pneumonia, septicemia, and meningitis. Primary Spn laboratory diagnostic identification methods include culture, antigen testing, single-plex real-time PCR, and syndromic PCR panels. However, each method lacks sensitivity, specificity, and/or cost efficiency. We developed and validated a quantitative, multiplex PCR assay using three Spn genomic targets ( lytA, piaB , and SP2020) for improved sensitivity and specificity to detect Spn in pleural fluid (PF), bronchoalveolar lavage (BAL), tracheal aspirate (TA), and upper respiratory (UR, research only) samples. Validation testing included analytical sensitivity (limit of detection), specimen storage, analytical specificity (cross-reactivity), and accuracy studies. Limit of detection is 500 genome copies/mL in lower respiratory samples and 100 copies/mL in upper respiratory specimens, with a quantification range of 1,000 to 10,000,000 copies/mL. Specimens can be stored frozen at least 60 days and Spn DNA is stable through three freeze-thaw cycles. No cross-reactivity was observed against 20 closely related microorganisms and/or microorganisms that can be detected in similar sample types, including Streptococcus pseudopneumoniae . In testing of residual clinical specimens, Spn was detected in 5 of 23 (21.7%) PF, 2 of 19 (10.5%) BAL, 1 of 20 (5.0%) TA, and 44 of 178 (24.7%) UR residual specimens. For accuracy studies, 98 specimens were tested and overall percent agreement with a qualitative, lytA -based comparator assay was 96.9% across all sample types. This multiplex, quantitative PCR assay is a sensitive and specific method for Spn detection in pediatric respiratory samples. IMPORTANCE Streptococcus pneumoniae ( Spn ) is the world’s leading cause of lower respiratory tract infection morbidity and mortality in children. However, current clinical microbiological methods have disadvantages. Spn can be difficult to grow in laboratory conditions if a patient is pre-treated, and Spn antigen testing has unclear clinical utility in children. Syndromic panel testing is less cost-effective than targeted PCR if clinical suspicion is high for a single pathogen. Also, such testing entails a full, expensive validation for each panel target if used for multiple respiratory sources. Therefore, better diagnostic modalities are needed. Our study validates a multiplex PCR assay with three genomic targets for semi-quantitative and quantitative Spn molecular detection from lower respiratory sources for clinical testing and from upper respiratory sources for research investigation.

Development of a real-time PCR assay for detection of hemp russet mite (Aculops cannabicola).

Of the many arthropod species affecting hemp (Cannabis sativa L.) cultivation in the United States, one species of particular importance is the hemp russet mite (Aculops cannabicola, HRM). Hemp russet mite is a microscopic arthropod which feeds on all parts of hemp plants. Due to its minute size, HRM can proliferate undetected for a long time, complicating management efforts and causing serious economic losses. DNA sequencing and PCR assays can facilitate accurate identification and early detection of HRM in infested-plants. Therefore, a real-time SYBR Green based species-specific PCR assay (quantitative PCR, qPCR) was developed for the identification of HRM DNA by amplification of a 104 bp Internal Transcribed Spacer 1 (ITS1) sequence. The detection limit was estimated to be approximately 48 copies of the HRM marker gene sequence. The real-time-PCR assay is rapid, detects all life stages of mite under 2 hours. A 10-fold serial dilution of the plasmid DNA containing the ITS1 insert were used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay showed a strong linear relationship with HRM DNA with R2 of 0.96. The assay was tested against several commonly found hemp pests including two-spotted spider mite and western flower thrips to determine specificity of the assay and to show that no non-target species DNA was amplified. The outcomes of this research will have important applications for agricultural biosecurity through accurate identification of HRM, early detection and timely deployment of management tactics to manage and prevent pest outbreaks.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples

Rapid Ohia Death (ROD) is caused by two pathogens, Ceratocystis lukuohia and Ceratocystis huliohia. The established species-specific real-time PCR assays targeting the single-copy cerato-platanin gene require DNA from between 2 and 16 spores per reaction for consistent detection, which is suitable for analysis of infected plant tissue but not sensitive enough to consistently detect low spore loads in environmental samples. Here, we present two redesigned qPCR assays targeting the first internal transcribed spacer (ITS) region of the multi-copy ribosomal DNA operon from the respective species, both capable of consistent detection of the pathogen at concentrations as low as 1 fg per reaction, less than the size of the haploid genome. Due to this increased sensitivity, these ITS qPCR assays are superior for analysis of DNA extracted from material collected in airborne particle samplers and from soil. Here, we demonstrate the utility of these qPCR assays for the characterization of windblown and soilborne dispersal of the pathogens, which is critical for the development of management practices that mitigate disease spread. [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, 2023.

Development of a Real-Time PCR Assay for Detection of Macrolide Resistance Mutations in Mycoplasma genitalium and Its Application for Epidemiological Surveillance in Russia.

A real-time PCR assay based on probe fluorescence quenching by a primer was developed and validated for detection of macrolide resistance (MR)-associated mutations in the 23S rRNA gene of Mycoplasma genitalium. The assay involves identification of any nucleotide substitutions at positions 2058, 2059, and 2611 of 23S rRNA (Escherichia coli numbering) by the probe-based melting curve analysis immediately after amplification and was capable of detecting target mutations in clinical specimens and spiked samples with 92% sensitivity and 100% specificity. We applied this new assay to assess the prevalence of MR-associated mutations in 949 nonduplicate urogenital samples positive for M. genitalium by routine diagnostic PCR, which were collected from symptomatic patients in five cities in the European part of Russia during the period 2009-2019. Forty-three (4.92%) samples revealed the presence of MR mutations, and no trend toward an increase in resistance prevalence was observed over the 10-year period of the study. The most commonly detected mutations were A2058G (26/43; 60.47%) and A2059G (13/43; 30.23%), while other mutations were rare: A2058T (3/43; 6.98%) and C2611T (1/43; 2.33%). The data obtained underline the need for regular epidemiological monitoring to ensure effective patient management, rational use of antibiotics, and prevention of further spread of resistance.

Validation of a Real-Time PCR Assay for Identification of Fresh and Processed Carica papaya Botanical Material: Using Synthetic DNA to Supplement Specificity Evaluation.

Several commercially important botanicals have a lack of diagnostic testing options that can quickly and unambiguously identify materials of different matrices. Real-time PCR can be a useful, orthogonal approach to identification for its exceptional specificity and sensitivity. Carica papaya L. is a species with a lack of available identification methods, and one which features two distinct commercially relevant matrices: fresh fruit and powdered fruit extract. In this study, we demonstrate the successful design and validation of a real-time PCR assay for detection of papaya DNA extracted from the two matrices. We also propose a technique that can be used during exclusivity panel construction, when genuine botanical samples are not available for certain species: substitution with synthetic DNA. We demonstrate the use of this material to complete a comprehensive specificity evaluation and confidently determine suitable Ct cutoff values. Further, we demonstrate how ddPCR can be used to determine the copy number of the target sequence in a set amount of genomic DNA, to which synthetic DNA samples can be corrected, and how it can verify specificity of the primers and probe. Through the presentation of successful assay validation for papaya detection, this work serves as a guideline for how to approach specificity evaluation when non-target botanical samples are difficult to obtain and otherwise may not have been included in the exclusivity panel.

Real-Time PCR Assay for detection and quantification of Leishmania: standardization, positive control, validation, and intra-laboratory assay

Real-Time PCR Assay for detection and quantification of Leishmania: standardization, positive control, validation, and intra-laboratory assay

Development and validation of a TaqMan® probe- based real-time PCR assay for detection of Ehrlichia canis

Ehrlichiosis is a potentially fatal zoonotic tick-borne disease, caused by a pleomorphic Gram-negative bacterium. It occurs worldwide and affects humans, domestic and wild animals. Dogs infected with Ehrlichia canis develop canine monocytic ehrlichiosis (CME), a significant infectious disease of canines. TaqMan® based real-time PCR assays to detect Ehrlichia spp. affecting dogs were developed and a real-time PCR assay specific for E. canis validated. The efficiency of the assay was 93% and the 95% limit of detection was 33 E. canis plasmid copies/µl of blood (95% confidence interval: 23 - 58). The assay was specific for E. canis when tested against other haemoparasites. Consistent repeatability was observed, with an inter-run standard deviation (SD) range between 0.33 and 1.29 and an intra-run SD range between 0.04 and 1.14. Field samples were tested in parallel by both the E. canis real-time PCR assay and a reverse line blot hybridization assay. The results were in agreement for the two assays, with an exception of two out of 121 samples. Bayesian latent class analysis was used to calculate a diagnostic sensitivity of the E. canis real-time PCR assay of 90% and a specificity of 92%. This assay is a sensitive and reliable molecular detection method for E. canis and will be a useful tool for early diagnosis and timely treatment for this haemoparasite.

Sensitive and Rapid Detection of Escherichia coli O157:H7 From Beef Samples Based on Recombinase Aided Amplification Assisted CRISPR/Cas12a System.

Escherichia coli O157:H7, being the cause of hemorrhagic colitis in humans, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive, and rapid E. coli O157:H7 detection method needs to be developed since the traditional detection methods are complex, costly, and time-consuming. In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive, and rapid nucleic acid detection of E. coli O157:H7 was introduced. First, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then a total of 34 bacterial strains were used for the specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157:H7 were prepared for the sensitivity test. Third, a real-time PCR assay for detection of the specific wzy gene of E. coli O157:H7 (FDA's Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out. The developed RAA-CRISPR/Cas12a method showed high specificity, and the detection could be completed within 30 min (after 4 h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4 × 102 CFU/mL and 7.5 × 10-4 ng/μL, respectively, which exhibited higher sensitivity than the RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157:H7 in ground beef samples could be positively detected after 4 h enrichment when the initial bacterial inoculum was 9.0 CFU/25 g. The detection results of the RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157:H7 from 93 local collected ground beef samples. The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157:H7 from ground beef samples. The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive, and accurate detection of E. coli O157:H7 in foods.

One-tube nested MGB Probe Real-time PCR assay for detection of Echinococcus multilocularis infection in plasma cell free DNA

IntroductionThe main objective of this study was to develop a One-tube nested MGB probe real-time PCR Assay for detecting Echinococcus multilocularis infection in human plasma cell free DNA (cfDNA). MethodscfDNA was extracted from 10 E.m.-infected patients using a NucleoSnap DNA Plasma Kit and characterized by genomic sequencing. We designed nested PCR primers and MGB probe for Echinococcus multilocularis detection. The specificity, sensitivity and reproducibility of this assay were analyzed, and its validity was confirmed in 13 early stage clinical samples. ResultsSeveral Echinococcus multilocularis-specific sequences were detected in the cfDNA of E.m.-infected patients, and CBLO020001206.1 was selected as the candidate sequence. We designed the primers and probe for the one tube nested real-time PCR. No cross-reactions with E.g. were observed. The detection limit was as low as 1 copy for Echinococcus multilocularis. The coefficients of variation were lower than 5% in intra- and inter-assays. 11 out of 13 patients were positive with nested MGB Probe PCR Assay and 3 patients were positive without outer primer in early stage Alveolar Echinococcosis pateints. ConclusionThe one-tube nested MGB probe real-time PCR assay is a simple, rapid, and cost-effective method for detection of Echinococcus multilocularis infection in patients’ Plasma DNA.

Development and validation of multiplex SYBR Green real-time PCR assays for detection and molecular surveillance of four tick-borne canine haemoparasites

Two multiplex SYBR Green based real-time PCR assays were standardized and evaluated to detect DNA from four canine haemoparasites (Babesia gibsoni, Babesia vogeli, Ehrlichia canis and Hepatozoon canis), along with internal controls from dogs from selected districts of Punjab state, India. Amplicons of 126 bp, 337 bp, 234 bp and 106 bp corresponding to B. gibsoni (18S rRNA gene), B. vogeli (18S rRNA gene), E. canis (virB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. Microscopic evaluation of 200 blood samples from dogs revealed the prevalence of B. gibsoni, E. canis and H. canis as 1.5%, 1.5% and 1.0%, respectively, while with the multiplex real-time PCR assays the values for B. gibsoni, B. vogeli, E. canis, and H. canis were 8.0%, 1.5%, 3.5% and 23.5%, respectively, with concurrent infections of B. gibsoni and H. canis (3.5%); E. canis and H. canis (2.0%) and B. gibsoni, B. vogeli, E. canis, and H. canis (0.5%). The diagnostic sensitivity of the multiplex real-time PCR assays with respect to microscopy in the detection of B. gibsoni, E. canis and H. canis was 100% while the specificity for B. gibsoni, B. vogeli, E. canis, and H. canis was 93%, 100%, 98% and 77%, respectively, revealing the respective strength of agreement as ″fair″, ″slight″, ″moderate″ and ″slight″ by kappa value statistics, and the data were statistically significant, for detection of B. gibsoni and E. canis infections, by Fisher's exact test. The analytical sensitivity of the multiplex PCR assays in detection of DNAs was 8.59 × 105 and 9.9 × 106 copies for B. vogeli and E. canis, respectively, and 1.15 × 106 and 3.41 × 105 copies for B. gibsoni and H. canis, respectively. Assessment of risk factors viz. age, sex, breed, season and locations showed no significant association with the prevalence of these haemoparasites except for B. vogeli, E. canis and H. canis where significant associations were found for location, age and breed, respectively by multiplex real-time PCR assays.

Surveillance of SARS-CoV-2 variants of concern by identification of single nucleotide polymorphisms in the spike protein by a multiplex real-time PCR

BackgroundAlthough active surveillance SARS-CoV-2 variants of concern (VOCs) is required for proper outbreak control measures, many lower income countries find it challenging to detect VOCs by carrying genomic sequencing alone, due to limited resources. MethodsVOCs can also be identified by the unique mutations in the spike protein by real-time PCR that detect these single nucleotide polymorphisms (SNPs). We used a multiplex, real-time PCR assay for detection of these SNPs for identification of the prevalence of different SARS-CoV-2 VOCs in 16/26 districts in Sri Lanka. ResultsOf the 664/934 that were subjected to the multiplex qRT-PCR, 638 (96.1 %) detected L452R and K417 in the channels and were identified as the delta variant. 25 samples (3.9 %) detected N501Y, with K417 were considered as the alpha variant. Of 10/16 districts in Sri Lanka, the delta variant was the only VOC detected. ConclusionsThis multiplex real-time qRT-PCR which identifies certain SNPs specific to the VOCs appears to be a fast, cheaper and less technically demanding method to generate data regarding the spread of different SARS-CoV-2 variants, and is a suitable method for lower income countries, to supplement the data generated by genomic sequencing.