PCR-based Assay For Detection Research Articles

Comparative Evaluation of AI-Based Multi-Spectral Imaging and PCR-Based Assays for Early Detection of Botrytis cinerea Infection on Pepper Plants

Pepper production is a critical component of the global agricultural economy, with exports reaching a remarkable $6.9B in 2023. This underscores the crop’s importance as a major economic driver of export revenue for producing nations. Botrytis cinerea, the causative agent of gray mold, significantly impacts crops like fruits and vegetables, including peppers. Early detection of this pathogen is crucial for a reduction in fungicide reliance and economic loss prevention. Traditionally, visual inspection has been a primary method for detection. However, symptoms often appear after the pathogen has begun to spread. This study employs the Deep Learning algorithm YOLO for single-class segmentation on plant images to extract spatial details of pepper leaves. The dataset included hyperspectral images at discrete wavelengths (460 nm, 540 nm, 640 nm, 775 nm, and 875 nm) from derived vegetation indices (CVI, GNDVI, NDVI, NPCI, and PSRI) and from RGB. At an Intersection over Union with a 0.5 threshold, the Mean Average Precision (mAP50) achieved by the leaf-segmentation solution YOLOv11-Small was 86.4%. The extracted leaf segments were processed by multiple Transformer models, each yielding a descriptor. These descriptors were combined in ensemble and classified into three distinct classes using a K-nearest neighbor, a Long Short-Term Memory (LSTM), and a ResNet solution. The Transformer models that comprised the best ensemble classifier were as follows: the Swin-L (P:4 × 4–W:12 × 12), the ViT-L (P:16 × 16), the VOLO (D:5), and the XCIT-L (L:24–P:16 × 16), with the LSTM-based classification solution on the RGB, CVI, GNDVI, NDVI, and PSRI image sets. The classifier achieved an overall accuracy of 87.42% with an F1-Score of 81.13%. The per-class F1-Scores for the three classes were 85.25%, 66.67%, and 78.26%, respectively. Moreover, for B. cinerea detection during the initial as well as quiescent stages of infection prior to symptom development, qPCR-based methods (RT-qPCR) were used for quantification of in planta fungal biomass and integrated with the findings from the AI approach to offer a comprehensive strategy. The study demonstrates early and accurate detection of B. cinerea on pepper plants by combining segmentation techniques with Transformer model descriptors, ensembled for classification. This approach marks a significant step forward in the detection and management of crop diseases, highlighting the potential to integrate such methods into in situ systems like mobile apps or robots.

Establishing methods to monitor H5N1 influenza virus in dairy cattle milk.

Highly Pathogenic Avian Influenza strain H5N1 has caused a multi-state outbreak among US dairy cattle, spreading across 15 states and infecting hundreds of herds since its onset. We rapidly developed and optimized PCR-based detection assays and sequencing protocols to support H5N1 molecular surveillance. Using 214 retail milk from 20 states for methods development, we found that H5N1 concentrations by digital PCR strongly correlated with qPCR cycle threshold (Ct) values, with dPCR exhibiting greater sensitivity. We also found that metagenomic sequencing after hybrid selection was best for higher concentration samples while amplicon sequencing performs best for lower concentrations. By establishing these methods, we were able to support the creation of a statewide surveillance program to test bulk milk samples monthly from all cattle dairy farms within Massachusetts, which remain negative to date. The methods, workflow, and recommendations described here provide a framework for others aiming to conduct H5N1 surveillance efforts.

Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat.

The traditional gold standard for detection of Salmonella in meat products is bacterial culture with enrichment. While this method is highly sensitive, it is slow and provides an incomplete assessment of isolate taxonomy in positive samples. This study presents a novel PCR based detection assay which amplifies the 16s-ITS-23s region which is an approximately 2500 base pair region of the larger ribosomal rrn operon. Intra-assay variation was assessed by splitting each biological sample into 3 technical replicates. Limits of detection (LOD) were assessed by utilizing a serial dilution of a pure culture of Salmonella enterica subsp. enterica serovar Heidelberg spiked into either sterile 1× PBS or 1× PBS rinsate of a Salmonella culture-negative chicken meat sample. Results indicate the 16s metabarcoding assay evaluated here could not be reliably used for the detection of Salmonella in adulterated retail meat samples as the LOD observed, 4.70 log colony forming units (CFU)/ml, is above the expected concentration of Salmonella in retail poultry meat samples which previous studies have shown range from under 1 to 2 log CFU/ml. However, due to greater taxonomic resolution afforded by using 16s long reads, the assay allowed alpha diversity assessment of the microbiome of raw poultry meat with the ability to assign taxonomy to the species and strain level for some amplicon sequence variants (ASV). This indicates this process may have value characterizing biodiversity and pathogen contamination of poultry samples in earlier steps of the poultry meat production process where bacterial contamination concentrations are likely to be higher.

Development, Optimization, and Validation of a Quantitative PCR Assay for Borrelia burgdorferi Detection in Tick, Wildlife, and Human Samples.

Tick-borne pathogens are growing in importance for human and veterinary research worldwide. We developed, optimized, and validated a reliable quantitative PCR (qPCR; real-time PCR) assay to assess Borrelia burgdorferi infection by targeting two B. burgdorferi genes, ospA and flaB. When assessing previously tested tick samples, its performance surpassed the nested PCR in efficiency, sensitivity, and specificity. Since the detection of Borrelia is more difficult in mammalian samples, the qPCR assay was also assessed using wildlife tissues. For wildlife samples, the sensitivity and specificity of ospA primers, with the incorporation of a pre-amplification step, was equivalent or superior to the nested PCR. For human samples, no primer set was successful with human tissue without culture, but we detected Borrelia with ospA and flaB primers in 50% of the Lyme culture samples, corresponding to 60% of the participants with a Lyme disease diagnosis or suspicion. The specificity of amplification was confirmed by Sanger sequencing. The healthy participant culture samples were negative. This PCR-based direct detection assay performs well for the detection of Borrelia in different biological samples. Advancements in detection methods lead to a better surveillance of Borrelia in vectors and hosts, and, ultimately, enhance human and animal health.

Role of Xpert PCR kit in assessing MRSA colonization in medical and surgical units of a tertiary care teaching hospital

Background and rationaleMethicillin resistant Staphylococcus aureus (MRSA) colonization increases the risk of MRSA infection. Detecting MRSA colonization can influence postoperative outcomes and prolong hospital stay. The conventional standard culture method for detecting MRSA colonization has limitations in terms of sensitivity and turnaround time. Hence, we sought out use of Xpert PCR kit for prompt evaluation of MRSA colonization to support MRSA prevention in a tertiary care hospital in Karachi, Pakistan.Materials and methodsDuring 1st April-31st December 2022, 290 nasal and skin swab samples were collected from 257 patients and processed using routine culture (as gold standard method) and PCR-based MRSA detection assay (MRSA Xpert).ResultsA total of two hundred and ninety (290) swab samples from 257 patients were obtained, 33 of which were paired. The overall prevalence of MRSA colonization was 12% by both methods, with 90% of cases classified as community-associated (CA-MRSA) whereas 10% as hospital-acquired (HA-MRSA). The colonized group showed a higher subsequent MRSA infection rate (11% vs. 3.5%) compared to the noncolonized group. Culture identified 11% of screening samples as MRSA positive, Xpert MRSA assay showed 100% sensitivity and 95% specificity. The cost of a single MRSA Xpert assay was $50 while MRSA culture cost around $7.50.ConclusionOur study findings suggest that the presence of MRSA colonization in our cohort of patients is consistent with the existing trends in hospital epidemiology. Both conventional culture and Xpert MRSA methods showed comparable efficacy for detection of MRSA colonization. Larger-scale studies are recommended to validate these findings conclusively.

Rapid detection of pathogenic E. coli based on CRISPR Cas system.

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.

Development of a PCR-based assay for specific and sensitive detection of Fusarium buharicum from infected okra plant.

Fusarium wilt, caused by the fungus Fusarium buharicum, is an emerging disease of okra in Japan. The disease was first reported in Japan in 2015, causing significant damage to okra seedlings. Due to the potential threat in okra cultivation, the development of an accurate detection method for F. buharicum is needed for the surveillance and management of the disease. In this study, we designed a primer set and developed conventional and nested PCR assays for the specific detection of F. buharicum in infected okra plants and contaminated soil, respectively. We compared the diversity of the translation elongation factor 1 alpha (EF-1α) gene of F. buharicum with 103 other fungal species/isolates to design a species-specific primer. This primer pair successfully amplified approximately 400 bp of PCR product that was only detected in the F. buharicum isolate, not in the other fungal isolates. The developed nested PCR method was highly sensitive and could detect the fungus from a 0.01 fg DNA sample. The primer successfully detected the pathogen in artificially infected plants and soil by conventional and nested PCR, respectively. This is the first report of the development of the F. buharicum-specific primer set and detection assays, which can be used for the specific and sensitive detection of F. buharicum in field samples and for taking early control measures.

Analysis of Parvovirus B19 persistence and reactivation in human heart layers

Heart disease is the leading cause of death worldwide. Myocarditis, or inflammation of the cardiac muscle, is estimated to cause up to 1.5 million cases annually, with viral infection being the most common disease culprit. Past studies have shown that Parvovirus B19 is routinely detected in endomyocardial biopsies. This virus has been linked to acute heart inflammation, which can cause cardiac muscle damage. However, because Parvovirus B19 can be found in the heart tissues in the absence of disease symptoms, it is unclear if the long-term presence of the virus contributes to, or initiates, heart disease. Here, we utilized a PCR-based detection assay to assess the presence of the B19V genome and its mRNA intermediates in human heart tissues. The analysis was carried out in three heart layers derived from one individual: epicardium, endocardium and myocardium. We showed the Parvovirus B19 genome presence variability in different heart layers. Similarly, viral transcriptional activity, assessed by the mRNA presence, was detected only in a few of the analyzed samples. Our results suggest that localized sites of Parvovirus B19 infection may exist within individual heart layers, which may have implication for the cardiac muscle inflammation.

Multiplex detection of bacterial pathogens by PCR/SERS assay.

Bacterial infections are a leading cause of death globally. The detection of DNA sequences correlated to the causative pathogen has become a vital tool in medical diagnostics. In practice, PCR-based assays for the simultaneous detection of multiple pathogens currently rely on probe-based quantitative strategies that require expensive equipment but have limited sensitivity or multiplexing capabilities. Hence, novel approaches to address the limitations of the current gold standard methods are still in high demand. In this study, we propose a simple multiplex PCR/SERS assay for the simultaneous detection of four bacterial pathogens, namely P. aeruginosa, S. aureus, S. epidermidis, and M. smegmatis. Wherein, specific primers for amplifying each target gDNA were applied, followed by applying SERS nanotags functionalized with complementary DNA probes and Raman reporters for specific identification of the target bacterial pathogens. The PCR/SERS assay showed high specificity and sensitivity for genotyping bacterial pathogen gDNA, whereby as few as 100 copies of the target gDNA could be detected. With high sensitivity and the convenience of standard PCR amplification, the proposed assay shows great potential for the sensitive detection of multiple pathogen infections to aid clinical decision-making.

A-294 Duplicate Testing of Samples Suspected of SARS-CoV-2 Infection Could Prevent Misdiagnosis of Covid-19

Abstract Background There is no doubt that the whole world has been shaken by the SARS-CoV-2 pandemic, which has affected both global health and the global economy. At the same time the importance of molecular methods for the accurate diagnosis of respiratory infections has been reinforced together with the need of testing methodologies that minimize the risk of misleading results. During the last three years of the pandemic, SARS-CoV-2 exhibited a tremendous adaptation capability through numerous mutations, leading to the appearance of the well-known Variants of Concern (VOCs) responsible of increased transmission rates. Therefore, the development of molecular methods capable of detecting all variants has become a challenge. To overcome this, potential genetic targets with low mutation rates have been identified, including ORF1ab, N gene, S gene and E gene. While Real-time PCR based detection assays of viral genomes are very accurate, testing naso/oropharyngeal samples using singlet analysis does not guarantee the error free detection of true positive results for SARS-CoV-2 infections. Furthermore, it has been documented that the probability of a false-negative testing results is influenced by a number of variables . Therefore, testing protocol adaptations, such as replicate testing, has been shown to efficiently reduce the probability of missing SARS-CoV-2 positive cases. The pilot study presented here investigates the outcome of testing 10 014 nasopharyngeal samples collected from patients with a suspected SARS-CoV-2 infection using two separate detection kits. In this study the error rate of single vs duplicate testing is discussed. Methods 10 014 nasopharyngeal swabs, collected from three different U.K. hospital trusts and Kent based care homes, were tested at the North Kent Pathology Service (NKPS). Each swab collected was placed in sterile transport media and stored at 4°C prior to viral genome isolation using the HigherPurity™ Viral RNA extraction kit. All collected specimens were processed within 24 h from collection. Total RNA isolated was immediately analyzed using two commercial CE-IVD kits: Vitassay qPCR SARS-CoV-2 and Primerdesign™ LTD, Coronavirus (COVID-19) Genesig Real-Time PCR assay. All samples were tested in duplicate. Results Following the testing and subsequent analysis, 491 samples (4.9%) were detected positive for SARS-CoV-2 using both assay kits. Of these, 302 samples (61.5%) were reported as duplicate positive (on both replicate 1 and 2) whereas 189 samples (38.5%) were detected positive on either replicate 1 or 2. In particular, 85 (17.3%) were detected positive on replicate 1 and 104 (21.2%) were detected positive on replicate 2. Conclusions Approximately 19% of tested individuals were detected as SARS-CoV-2 positive based on a positive Real-time PCR result in one of the two reactions. This percentage represents the potential SARS-CoV-2 infections that can be missed when a single reaction Real-time PCR testing is employed for infection detection in the wider population, and equates to 940 individuals out of 100 000 potentially being falsely confirmed as SARS-CoV-2 negative The authors believe that in the presence of fast transmitting variants this is a significant number of false negative tests and should be taken into consideration when testing protocols are developed and disease modelling is employed.

Conditional Forest Models Built Using Metagenomic Data Accurately Predicted Salmonella Contamination in Northeastern Streams.

The use of water contaminated with Salmonella for produce production contributes to foodborne disease burden. To reduce human health risks, there is a need for novel, targeted approaches for assessing the pathogen status of agricultural water. We investigated the utility of water microbiome data for predicting Salmonella contamination of streams used to source water for produce production. Grab samples were collected from 60 New York streams in 2018 and tested for Salmonella. Separately, DNA was extracted from the samples and used for Illumina shotgun metagenomic sequencing. Reads were trimmed and used to assign taxonomy with Kraken2. Conditional forest (CF), regularized random forest (RRF), and support vector machine (SVM) models were implemented to predict Salmonella contamination. Model performance was assessed using 10-fold cross-validation repeated 10 times to quantify area under the curve (AUC) and Kappa score. CF models outperformed the other two algorithms based on AUC (0.86, CF; 0.81, RRF; 0.65, SVM) and Kappa score (0.53, CF; 0.41, RRF; 0.12, SVM). The taxa that were most informative for accurately predicting Salmonella contamination based on CF were compared to taxa identified by ALDEx2 as being differentially abundant between Salmonella-positive and -negative samples. CF and differential abundance tests both identified Aeromonas salmonicida (variable importance [VI] = 0.012) and Aeromonas sp. strain CA23 (VI = 0.025) as the two most informative taxa for predicting Salmonella contamination. Our findings suggest that microbiome-based models may provide an alternative to or complement existing water monitoring strategies. Similarly, the informative taxa identified in this study warrant further investigation as potential indicators of Salmonella contamination of agricultural water. IMPORTANCE Understanding the associations between surface water microbiome composition and the presence of foodborne pathogens, such as Salmonella, can facilitate the identification of novel indicators of Salmonella contamination. This study assessed the utility of microbiome data and three machine learning algorithms for predicting Salmonella contamination of Northeastern streams. The research reported here both expanded the knowledge on the microbiome composition of surface waters and identified putative novel indicators (i.e., Aeromonas species) for Salmonella in Northeastern streams. These putative indicators warrant further research to assess whether they are consistent indicators of Salmonella contamination across regions, waterways, and years not represented in the data set used in this study. Validated indicators identified using microbiome data may be used as targets in the development of rapid (e.g., PCR-based) detection assays for the assessment of microbial safety of agricultural surface waters.

Investigation of respiratory disease outbreaks of poultry in Bangladesh using two real-time PCR-based simultaneous detection assays.

For rapid and sensitive pathogen screening from field outbreaks, molecular techniques such as qPCR-based simultaneous detections are efficient. Respiratory diseases are the most detrimental diseases to the poultry industry and need to be addressed because of their major economic losses. In the current study, we have applied two different detection assays: one for simultaneous detection of avian influenza virus (AIV; M gene) and subtyping (H5, N1, H9, N2) using TaqMan probe chemistry (TaqMan multitarget) and another for simultaneous detection of Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) using SYBR Green chemistry (SYBR Green multitarget). Two individual qPCRs were conducted for the detection of four pathogens. Surveillance of tissue (n = 158) and oropharyngeal swab (206) samples from multiple poultry flocks during the years April 2020-July 2022 applying the TaqMan and SYBR Green multitarget qPCRs revealed that 48.9% of samples were positive for respiratory infections, of which 17.2% were positive for NDV, 25.5% were positive for AIV, 9.9% were positive for IBV, and only a single positive (0.3%) for ILTV. Among the AIV, 35% were highly pathogenic subtype H5N1 and 65% were low pathogenic subtype H9N2. Co-infections of 2-3 respiratory viruses were also accurately detected. Respiratory viral pathogens are quite common in Bangladeshi poultry and can be successfully detected using multitarget simultaneous real-time quantitative polymerase chain reaction (RT-qPCR) assays like those adopted in the current study. Increased mass surveillance, along with the molecular characterization of the circulating respiratory viruses, is crucial to control the epidemic and subsequently save the Bangladeshi poultry industry.

Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay

A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP). Upon incubation with serum or DBS samples, the bi- or multivalency of the antibodies (IgG, IgA or IgM) brings pairs of viral proteins with their conjugated oligonucleotides in proximity, allowing the antibodies to be detected by a modified proximity extension assay (PEA). Anti-S1 and anti-NP antibodies could be detected simultaneously from one incubation reaction. This Antibody PEA (AbPEA) test uses only 1 µl of neat or up to 100,000-fold diluted serum or one ø1.2 mm disc cut from a DBS. All 100 investigated sera and 21 DBS collected prior to the COVID-19 outbreak were negative, demonstrating a 100% specificity. The area under the curve, as evaluated by Receiver Operating Characteristic (ROC) analysis reached 0.998 (95%CI: 0.993–1) for samples taken from 11 days after symptoms onset. The kinetics of antibody responses were monitored after a first and second vaccination using serially collected DBS from 14 individuals. AbPEA offers highly specific and sensitive solution-phase antibody detection without requirement for secondary antibodies, no elution step when using DBS sample in a simple procedure that lends itself to multiplex survey of antibody responses.

A one-step multiplex PCR-based assay for simultaneous detection and classification of virulence factors to identify five diarrheagenic E. coli pathotypes

Human diarrhea-causing strains of Escherichia coli are referred to as diarrheagenic E. coli (DEC). DEC can be divided into five main categories based on distinct epidemiological and clinical features, specific virulence determinants, and association with certain serotypes. In the present study, a simple and rapid one-step single reaction multiplex PCR (mPCR) assay was developed for the simultaneous identification and differentiation of five currently established DEC pathotypes causing gastrointestinal diseases. The mPCR incorporated 10 primer pairs to amplify 10 virulence genes specific to the different pathotypes (i.e., stx1 and stx2 for EHEC, elt and sth for ETEC, eaeA and bfpA for EPEC, aggR and astA for EAEC, and ipaH and invE for EIEC) and to generate DNA fragments of sufficiently different sizes to be unequivocally resolved. All strains were detected at concentrations ranging from 104 to 107 CFU/mL. To demonstrate the utility of the mPCR assay, 236 clinically isolated strains of DEC from two hospitals were successfully categorized. One-step mPCR technique reduced the cost and effort involved in the identification of various virulence factors in DEC. Thus, we demonstrated that the newly developed mPCR assay has the potential to be introduced as a diagnostic tool that can be utilized for the detection of DEC as an additional check in clinical laboratories and for confirmation in health and environment institutes, health centers, and reference laboratories.

Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

Development of PCR-Based Assays for Rapid and Reliable Detection and Identification of Canker-Causing Pathogens from Symptomatic Almond Trees.

Trunk and scaffold canker diseases (TSCDs) of almond cause significant yield and tree losses and reduce the lifespan of orchards. In California, several pathogens cause TSCDs, including Botryosphaeriaceae, Ceratocystis destructans, Eutypa lata, Collophorina hispanica, Pallidophorina paarla, Cytospora, Diaporthe, and Phytophthora spp. Field diagnosis of TSCDs is challenging because symptom delineation among the diseases is not clear. Accurate diagnosis of the causal species requires detailed examination of symptoms and subsequent isolation on medium and identification using morphological criteria and subsequent confirmation using molecular tools. The process is time-consuming and difficult, particularly as morphological characteristics are variable and overlap among species. To facilitate diagnosis of TSCD, we developed PCR assays using 23 species-specific primers designed by exploiting sequence differences in the translation elongation factor, β-tubulin, or internal transcribed spacer gene. Using genomic DNA from pure cultures of each fungal and oomycete species, each primer pair successfully amplified a single DNA fragment from the target pathogen but not from selected nontarget pathogens or common endophytes. Although 10-fold serial dilution of fungal DNA extracted from either pure cultures or infected wood samples detected as little as 0.1 pg of DNA sample, consistent detection required 10 ng of pathogen DNA from mycelial samples or from wood chips or drill shavings from artificially or naturally infected almond wood samples with visible symptoms. The new PCR assay represents an improved tool for diagnostic laboratories and will be critical to implement effective disease surveillance and control measures.

Development of a sensitive and single-step PCR-based assay for detection of sandal spike phytoplasma

Development of a sensitive and single-step PCR-based assay for detection of sandal spike phytoplasma

Abstract 5996: Single tube PCR-based NGS assay for detection of multiple gene fusions from cell free total nucleic acid

Abstract Introduction: Recently, liquid biopsy has begun to be adopted for treatment selection and drug-resistance assessment for cancers. However, currently liquid biopsy is mostly focused on analyzing cell-free DNA (cfDNA). Though analyzing RNA provides more accurate analysis for functional gene fusions, little is known about the stability and availability of cfRNA (cell free RNA). Here we report our one-tube robust cfTNA (cell free total nucleic acids) assay to analyze ctDNA and ctRNA simultaneously. Methods: We assessed Pillar’s cell free TNA panel performance on 2 confirmed fusion positive and 5 fusion negative samples that were serially diluted to obtain a total of 88 samples including replicates. An input of 10ng cfTNA was used for fusion positive samples with fusion frequency ranging from 0.2% to 20%. A range of 5 to 30ng input was used for fusion negative samples. The amount of input cfRNA was estimated as equal to the cfDNA input. Total nucleic acids were extracted and cfRNA was reverse transcribed. Amplicons targeted the specific breakpoints of 36 known fusion transcripts. Fusion positive samples were diluted for library prep to achieve fusion frequency of 0.5%, 1%, 4%, 5% and 20%. Novaseq was used for sequencing with an average depth of 200,000X and downsampled by 25%. Pillar Variant Analysis Toolkit (PiVAT®) was used to map and call fusions. Results: Among dilutions of >=4%, PiVAT achieved >=99% sensitivity and >=99.9% specificity in calling RNA fusions from 10ng input. Even at extremely low dilutions of <=1%, PiVAT achieved >= 50% sensitivity. Conclusion: Pillar’s cell free TNA panel offers a simple, rapid, and reliable workflow that enables detection of a broad range of clinically relevant gene fusions. Detection of fusion events in cell-free samples from noninvasive liquid biopsies represents an important step forward in cancer diagnosis and monitoring. Citation Format: Tejashree Modak, Jilong Li, Sean Polvino, Yue Ke, Zhaohui Wang, Alex V. Kotlar. Single tube PCR-based NGS assay for detection of multiple gene fusions from cell free total nucleic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5996.

Comparison of 3 PCR-based assays for microsatellite instability detection in formalin-fixed paraffin-embedded tissues of patients with colorectal cancer.

e15042 Background: Routine assessment of microsatellite instability (MSI) status in colorectal cancer (CRC) patients can help with initial screening for Lynch syndrome, prediction of tumor prognosis and selection of patients for immunotherapy. Immunohistochemistry (IHC) and PCR-based approaches represent the current clinical laboratory standard for the evaluation of MSI status. Both Bethesda and Promega marker panels have been used widely in the world and the Bethesda panel and the six mononucleotides panel (NR21/BAT25/NR24/NR27/MONO27/BAT26) have been approved by the National Medical Products Administration (NMPA) in China. The Idylla MSI Test, a fully automated system based on real-time PCR, which incorporates 7 novel MSI loci (ACVR2A/BTBD7/DIDO1/MRE11/RYR3/SEC31A/SULF2) can perform MSI analysis within 150min using 1-3 tissue slides. The aim of this study was to evaluate the performance of the three 3 PCR-based assays for MSI detection including Idylla MSI Test, Bethesda and that six mononucleotides panel. Methods: One hundred and fifty-three formalin-fixed paraffin-embedded (FFPE) tumor tissues from patients with CRC were collected and their MSI status were analyzed using 3 PCR-based assays, respectively, i.e. the Idylla MSI, “2B3D” NCI panel and the six mononucleotides panel. All samples were previously evaluated by IHC method. Results: In the all 153 patients, IHC identified 27 cases to be MMR-deficient (dMMR), and 126 to be MMR-proficient (pMMR). Using IHC as a reference, the Idylla MSI had a higher consistency that showed overall agreement, sensitivity and specificity as 93.46%, 92.89% and 93.65%, respectively, while the corresponding values of the six mononucleotides panel were 84.31%, 92.59% and 82.54% respectively, and the “2B3D” NCI panel showed a sensitivity of 92.16%, a specificity of 88.89% and an overall concordance of 92.86%. The PCR-based approaches identified 11 more MSI-H cases than IHC while 22 dMMR cases identified by IHC were detected by all the 3 assays, 2 dMMR by 2 assays, 1 dMMR by one assay only and 1 dMMR by neither assays above. Moreover, the Idylla MSI showed advantages in ease of use, turnaround time (TAT) and small dose of tissue input. Conclusions: Our findings support that the Idylla MSI assay provides a rapid, reliable, and ease-to-use solution to MSI detection in Chinese CRC patients with high sensitivity and specificity. The inconsistent cases between IHC and PCR-based approaches need a further analysis.

Clinical Validation of the Fully Automated NeuMoDx HPV Assay for Cervical Cancer Screening.

The NeuMoDx HPV assay is a novel fully automated, real-time PCR-based assay for the qualitative detection of high-risk human papillomavirus (HPV) DNA in cervical specimens. The assay specifically identifies HPV16 and HPV18 and concurrently detects 13 other high-risk HPV types at clinically relevant infection levels. Following the international guidelines, the clinical performance of the NeuMoDx HPV assay for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) against the reference standard Hybrid Capture 2, as well as intra- and inter-laboratory reproducibility were assessed on PreservCyt samples. The clinical accuracy of the assay was additionally evaluated against the clinically validated Alinity m HR HPV and COBAS 4800 HPV Test on PreservCyt samples, and against the clinically validated HPV-Risk assay on SurePath samples. The NeuMoDx HPV assay performance for CIN2+ was non-inferior to the reference methods on both sample types (all p < 0.05), and showed excellent intra- and inter-laboratory reproducibility (95.7%; 95% CI: 93.9–97.3; kappa value 0.90 (95% CI: 0.86–0.94); and 94.5%; 95% CI: 92.6–96.2; kappa value 0.87 (95% CI: 0.82–0.92), respectively). In conclusion, the NeuMoDx HPV assay meets international guideline criteria for cross-sectional accuracy and reproducibility, and performs equally well on cervical screening specimens collected in two widely used collection media. The NeuMoDx HPV assay fulfils the requirements to be used for primary cervical screening.