Multiplex Real-time PCR Assay Research Articles

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 mM) or rotenone (0.2-50 mM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.

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

Shiga toxin-producing Escherichia coli (STEC) are food-borne 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 Stx2 g). An additional nine subtypes have been reported and described (Stx1e, Stx2 h 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 application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses.

Porcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses. A multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay. This multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/μL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively. The multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations.

Development of a multiplex real-time PCR assay for simultaneous detection of common colistin and carbapenemase genes

Carbapenem and colistin are often used as last-resort treatment for Gram-negative multi-drug resistant (MDR) bacteria. Nevertheless, co-resistance of these drugs is threatening the global healthcare system. Rapid and accurate detection of carbapenem and colistin resistant bacteria is critical for adequate antibiotic therapy and infection control, particularly in the context of an outbreak. The presence of blaNDM, blaKPC, blaIMP-1 and blaOXA-48 is responsible for greater than 95% phenotypic resistance in carbapenem-resistant Enterobacteriaceae, while mcr-1 is the most prevalent and well disseminated of all mcr genes in colistin-resistant strains. In this study, we aim to develop a multiplex real time-PCR assay for simultaneous detection of the five genes blaNDM, blaKPC, blaIMP-1, blaOXA-48 and mcr-1. The melting curve-based multiplex real time PCR assay was established with the dissociation temperature range extended from 76°C to 87°C. The whole process is completed within one hour and half, allowing rapid screening of the five genes in cultured bacteria samples with a limit of detection of 10 CFU/ml. The proposed multiplex real-time PCR assay is a robust, reliable and rapid method for the detection of bacterial strains carrying blaOXA-48, blaIMP, blaNDM, blaKPC and mcr-1 gene individually or in cocktail of genes. This assay will be a valuable tool for surveillance and monitoring of MDR bacteria additionally resistant to either carbapenem or colistin or both drugs.

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

A novel multiplex real-time PCR assay for the detection of cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2 and strategies for application to blood screening

A multiplex real-time PCR has been developed to simultaneously detect transfusion-transmissible pathogens cytomegalovirus, Epstein-Barr virus and herpes simplex virus, as well as to provide sample quality testing, for the conserved regions of the cytomegalovirus UL123 gene, the Epstein-Barr virus BKRF1 gene, and the herpes simplex virus 1/2 UL30 gene, tested on 500 blood donors and 320 transfusion recipients. The laboratory sensitivities for all 3 pathogens were 100 copies/μL. Compared to the commercial real-time PCR reference kit, the multiplex real-time PCR assay for the detection of CMV, EBV and HSV presented 100% consistency. In 820 whole blood samples, the multiplex real-time PCR assay identified 34 (4.15%) positive for CMV DNA, 15 (1.83%) positive for EBV DNA, and 6 (0.73%) positive for HSV DNA. For blood transfusions in high-risk groups, whole blood herpes virus test should be included in the spectrum of pathogen testing for blood donors and recipients.

Enhancing enteric pathogen detection: implementation and impact of multiplex PCR for improved diagnosis and surveillance

BackgroundSyndromic surveillance of acute gastroenteritis plays a significant role in the diagnosis and management of gastrointestinal infections that are responsible for a substantial number of deaths globally, especially in developing countries. In Lebanon, there is a lack of national surveillance for acute gastroenteritis, and limited data exists regarding the prevalence of pathogens causing diarrhea. The one-year study aims to investigate the epidemiology of common gastrointestinal pathogens and compare our findings with causative agents of diarrhea reported by our study collaborative centers.MethodsA multicenter, cross-sectional study was conducted over a one-year period. A total of 271 samples were obtained from outpatients and inpatients presenting with symptoms of acute gastroenteritis at various healthcare facilities. The samples were then analyzed using Allplex gastrointestinal assay that identifies a panel of enteric pathogens.ResultsOverall, enteropathogens were detected in 71% of the enrolled cases, 46% of those were identified in patients as single and 54% as mixed infections. Bacteria were observed in 48%, parasites in 12% and viruses in 11%. Bacterial infections were the most prevalent in all age groups. Enteroaggregative E. coli (26.5%), Enterotoxigenic E. coli (23.2%) and Enteropathogenic E. coli (20.3%) were the most frequently identified followed by Blastocystis hominis (15.5%) and Rotavirus (7.7%). Highest hospitalization rate occurred with rotavirus (63%), Enterotoxigenic E. coli (50%), Blastocystis hominis (45%) and Enteropathogenic E. coli (43%). Enteric pathogens were prevalent during summer, fall and winter seasons.ConclusionsThe adoption of multiplex real-time PCR assays in the diagnosis of gastrointestinal infections has identified gaps and improved the rates of detection for multiple pathogens. Our findings highlight the importance of conducting comprehensive surveillance to monitor enteric infections. The implementation of a syndromic testing panel can therefore provide healthcare professionals with timely and accurate information for more effective treatment and public health interventions.

Molecular characterization of human bocavirus in municipal wastewaters using amplicon target sequencing

Human bocavirus (HBoV) is an emerging health concern worldwide, associated with range of clinical manifestations, including gastroenteritis and respiratory infections. Therefore, it is crucial to comprehend and minimize their prevalence in different systems. In this study, we conducted regular sampling throughout the year in two different sizes and work processes of wastewater treatment plants (WWTPs) in Tianjin, China. Our objective was to investigate the occurrence, prevalence, and endurance of HBoV in wastewater, while also evaluating the efficacy of amplicon target sequencing in directly detecting HBoV in wastewater. At two WWTPs, HBoV2 (45.51 %—45.67 %) and HBoV3 (38.30 %—40.25 %) were the most common genotypes identified, and the mean concentration range of HBoV was 2.54—7.40 log10 equivalent copies/l as determined by multiplex real-time quantitative PCR assay. A positive rate of HBoV was found in 96.6 % (29/30) samples of A-WWTP, and 96.6 % (26/27) samples of B-WWTP. The phylogenetic analysis indicated that the nucleotide similarity between the HBoV DNA sequences to the reference HBoV sequences published globally ranged from 90.14 %—100 %. A significant variation in the read abundance of HBoV2 and HBoV3 in two wastewater treatment plants (p < 0.05) was detected, specifically in the Winter and Summer seasons. The findings revealed a strong correlation between the genotypes detected in wastewater and the clinical data across various regions in China. In addition, it is worth mentioning that HBoV4 was exclusively detected in wastewater and not found in the clinical samples from patients. This study highlights the high prevalence of human bocavirus in municipal wastewater. This finding illustrates that amplicon target sequencing can amplify a wide variety of viruses, enabling the identification of newly discovered viruses.

Rift Valley Fever virus M and L genome segment detection: a comparison of field-deployable reverse transcription insulated isothermal PCR (RT-iiPCR) and laboratory-based multiplex reverse transcription real-time PCR.

The content of this manuscript is of interest to the diverse readership of the Journal of Clinical Microbiology, including research scientists, diagnosticians, healthcare professionals, and policymakers. Rift Valley Fever virus (RVFV) is a zoonotic mosquito-borne pathogen that causes major agricultural and public health problems. Current and most sensitive diagnostic approaches that are molecular-based are performed in highly specialized molecular diagnostic laboratories. To address diagnostic needs, we developed a novel, rapid, and sensitive molecular method using a portable PCR machine, POCKIT, capable of immediate deployment at sites of suspected outbreaks. Here, we demonstrate that field-deployable RVFV detection can provide reliable, sensitive, and specific point-of-need viral RNA detection that could be used for diagnostic investigations and epidemiological studies, and can be performed in the field.

In-house RT-PCR detection of hepatitis A virus in human plasma pools used for the manufacture of medicinal products

Hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is transmitted through the fecal-oral route. However, HAV can also be transmitted by blood-derived products. This is due to the fact that viremia occurs during the asymptomatic phase of HAV infection, enabling infected blood or plasma donations to occur. Viral inactivation/removal steps are included during manufacturing of plasmaderived products. However, HAV is a small non-enveloped virus very difficult to remove with traditional viral inactivation procedures. To accomplish European guidelines for pooled human plasma (treated for virus inactivation), plasma manufacturers have been implementing HAV nucleic acid test (NAT) screening on plasma pools. In this study, we validate an in-house multiplex reverse-transcription real-time PCR (RT-PCR) assay targeting HAV RNA and an internal control with hydrolysis probes for amplicon detection. The HAV RNA test was validated by assessing limit of detection, robustness, sensitivity and specificity according to European Pharmacopoeia (Ph. Eur.) guidelines. Our assay is able to detect 100 IU/mL of all human HAV genotypes that have been described so far. The multiplex assay shows remarkable sensitivity with a 95% lower limit of detection of 5.2 IU/mL. Also, our HAV test shows good robustness, precision, and specificity. We conclude that our assay broadly meets the requirements for its purpose. The implementation of this test in the production process of plasma-derived products will increase their safety.

A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses.

Calf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high. To address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes. This method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/μL, 96.0 copies/μL, 12.8 copies/μL, 16.4 copies/μL, 18.2 copies/μL, and 65.3 copies/μL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence. Additionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.

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.

Development of a multiplex real time-PCR assay to estimate the distribution of the genus Silurus in Lake Biwa using environmental DNA

Development of a multiplex real time-PCR assay to estimate the distribution of the genus Silurus in Lake Biwa using environmental DNA

Establishment and application of a multiplex real-time PCR assay coupled with propidium monoazide for the simultaneous detection of viable Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus

A propidium monoazide (PMA) - multiplex real-time PCR (mRT-PCR) assay was established for the simultaneous detection of viable Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus. According to the unique sequences of the target genes identified by our laboratory for the above pathogenic bacteria, an efficient and specific mRT-PCR method based on TaqMan probes was developed, and then PMA was introduced into the mRT-PCR system. The optimization of the PMA treatment conditions successfully achieves the purpose of distinguishing viable and dead bacteria in the cell suspension, and effectively eliminates false positive results caused by dead cells. The method's specificity was verified by 54 strains, exhibiting 100% specificity without cross-reactivity with other pathogens. Furthermore, the developed method could accurately detect the target bacteria in the presence of non-target bacteria at a concentration range of 100-107 CFU/mL, demonstrating excellent anti-interference ability. The detection limits of V. parahaemolyticus, V. vulnificus, and V. alginolyticus were 2.0 × 101 CFU/mL, 3.2 × 101 CFU/mL, and 3.75 × 101 CFU/mL, respectively. To reflect the practicability of the method, the established method was developed as a detection kit, and the evaluation results showed that the kit has good stability and good reproducibility. The applicability of this kit was satisfying after evaluating in artificially spiked aquatic products (shrimp, yellow croakers and oysters) with recoveries ranging from 90.22% to 109.35%. The PMA-mRT-PCR detection assay is a feasible method for the rapid, sensitive, and quantitative detection of bacterial pathogens. And the novel kit developed based on this method has significant application value for the large-scale screening and identification of harmful Vibrios in food matrices.

Development of an ARMS multiplex real-time PCR assay for the detection of HLA-B*13:01 genotype by detecting highly specific SNPs.

HLA-B*13:01 was strongly associated with Dapsone Hypersensitivity Syndrome (DHS). This study aimed to develop and validate a rapid and economical method for HLA-B*13:01 genotyping. Two tubes multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*13:01 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. The accuracy of the assay was 100% for HLA-B*13:01 genotyping. The detection limit of the new method was 0.025 ng DNA. The positive rate of HLA-B*13:01 in the Bouyei (20%, n = 50) populations was significantly higher than that in the Uighur population (4%, n = 100), Han (4.5%, n = 200), and Tibetan (1%, n = 100) ( P < 0.05). The proposed method is rapid and reliable for HLA-B*13:01 screening in a clinical setting.

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 Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

Multiplex high resolution melt curve real-time PCR for detection of Shiga-toxin producing and blaCTX-M-harboring E. coli in beef products

This study aimed to develop a rapid multiplex high resolution melt-curve (HRM) qPCR with an internal amplification control (IAC) for the simultaneous detection of Shiga-toxin producing Escherichia coli (STEC) and blaCTX-M-carrying E. coli in beef. Specific primer pairs targeting eaeA, uidA, stx1, stx2 and blaCTX-M genes were designed. The specificity of the optimized assay was validated using genomic DNA from Gram-positive, Gram-negative, and STEC (non-O157 and O157:H7) strains. The accuracy of the assay for detecting blaCTX-M was confirmed using genomic DNA from bacteria previously reported to carry this gene. Beef products were inoculated with a cocktail of E. coli O157:H7 and E. coli M-1, an isolate reported to harbor blaCTX-M, at concentrations of 10, 100, and 1000 CFU/mL. Among non-STEC DNA samples (n = 45) tested, no target genes were detected in 32 isolates. However, the assay was able to detect uidA and blaCTX-M in seven and six bacterial isolates, respectively. Among the STEC bacterial isolates (n = 57), at least two or more target genes were detected in 50 STEC isolates. All isolates carried the eaeA gene, 55 had stx1, 26 had stx2, and 26 had both stx genes. The uidA gene was detected in all non-O157:H7 isolates, whereas no E. coli O157:H7 isolates was found to be positive for uidA. The blaCTX-M gene was not detected in any of the STEC isolates tested but it was detected in 20 of 23 DNA samples from isolates previously verified as positive for the gene, thus demonstrating the technique's accuracy. Enrichment times ranging from 4 to 8 h were required to achieve a detection limit of 10 CFU/mL in 325 g of inoculated beef. The multiplex HRM real-time PCR assay can be used as a rapid, yet specific, tool for routine examination of STEC and blaCTX-M-harboring bacteria that are present in low concentrations in beef products.

B-237 Analytical and Clinical Performance Evaluation of the Panther Fusion® GI Parasite Assay (In Development)

Abstract Background Acute diarrhea from gastrointestinal (GI) infections is the leading cause of outpatient visits, hospitalizations, and loss of quality of life, with an estimated global impact of 500 million illnesses and 230 000 deaths annually. Most GI infections from bacteria, viruses, and parasites present similar symptoms, but successful treatment is dependent on accurate pathogen identification. For identification of parasites, microscopic testing is often used, but is laborious and often results in inconclusive or inaccurate diagnoses. Clinicians now rely on rapid and accurate molecular diagnostics to correctly identify the causative organism, which leads to optimal infection control and appropriate treatment. The Panther Fusion (PF) GI Parasite Assay is a multiplex qualitative real-time PCR assay that addresses the need for accurate and sensitive identification and detection of the most common parasites that are known to cause gastroenteritis: Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis. Methods Stool specimens collected in Cary-Blair (CB) are transferred to the AptimaTM Multitest collection tube (Hologic). Multitest tubes are loaded on Panther Fusion where nucleic acid isolation, amplification, and detection are performed. Analytical sensitivity was assessed using cell suspension of Cryptosporidium, Giardia or E. histolytica spiked in negative CB Stool (CBS) matrix and the limit of detection (LoD) was determined using probit. Since Cyclospora cayetanensis cannot be cultured, in-vitro transcribed RNA (IVT) was spiked in CBS matrix for LoD evaluation. Inclusivity was evaluated using cell suspensions for 20 Giardia and 15 E. histolytica strains spiked in CBS matrix. Cryptosporidium inclusivity was evaluated with synthetic nucleic acid. Specificity was evaluated by spiking cells suspensions or synthetic nucleic acid of various microbes in CBS in the presence or absence of GI targets. For clinical performance, 119 archived frozen stool specimens in CB and 40 contrived specimens were tested on Film Array GI Panel (BioFire) and PF GI Parasite Assay and results were compared. Results The LoD for Cryptosporidium, Giardia and E. histolytica was determined to be &amp;lt; 1 cell/mL in the Aptima Multitest tube. For C. cayetanensis, the LoD was found to be 1032 copies/mL. The PF GI Parasite Assay detected 20 strains of Giardia lamblia and 15 E. histolytica strains. In-silico and initial testing of synthetic nucleic acid demonstrated detection of 10 Cryptosporidium species. No cross-reactivity to closely related organisms or other microorganisms commonly found in the gastrointestinal tract was observed, except for cross-reactivity to Entamoeba nuttalli, which rarely infects humans. In testing of clinical remnant specimens, the PF GI Parasite Assay had a 97% PPA (35/36) for Cryptosporidium, 100% PPA (58/58) for Giardia lamblia, 100% PPA (42/42) for E. histolytica (included 40 contrived specimens) and 100% PPA (8/8) for Cyclospora with the Film Array GI Panel. Conclusion The PF GI Parasite Assay demonstrated excellent analytical and clinical performance in the detection and differentiation of Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis. Together with its companion Panther Fusion assays that detect bacterial and viral GI pathogens (also in development), this assay will provide a sensitive and accurate molecular tool for the automated detection of parasites in human stool.