Polymerase Chain Reaction Amplification Research Articles

Assessment of in vitro dynamics of pathogenic environmental Acanthamoeba T4 and T9 genotypes isolated from three recreational lakes in Klang Valley, Malaysia over the HaCaT cell monolayer

ABSTRACT Free-living amoebae of the genus Acanthamoeba are causative agents of keratitis and amoebic encephalitis. They are widely found in various ecological environments. Therefore, the present study brings results that can help to better understand the genotypes of the environmental isolates and their pathogenicity. This study procured 26 Acanthamoeba isolates from three recreational lakes in 2022. Polymerase chain reaction amplification was performed on positive Acanthamoeba samples. The thermotolerance, osmotolerance, and cytopathogenicity in human keratinocyte (HaCaT) cells of the samples were also evaluated. The phylogenetic analysis demonstrated that 12 isolates were of genotype T4, two (T9), six (T17), four (T8), and one each from T5 and T11. The thermo- and osmotolerance assays indicated that eight Acanthamoeba samples were potentially pathogenic. Two T4 and one T9 genotype also recorded 33-, 42-, and 133-kDa serine-type proteases, respectively. The HaCaT cell monolayer revealed that three T4 and one T9 samples achieved cytopathic effects within the 50–100% range, hence significantly cytotoxic. The lactate dehydrogenase secretion results demonstrated that three (T4) and one (T9) sample exhibited exceptional toxicity (over 40%) compared to the other samples. The responses of Acanthamoeba members with similar genotypes to pathogenicity indicator assays varied considerably, rendering correlation of pathogenicity with specific genotypes challenging.

Analysis of Methylation of Tumor-suppressive miRNAs and KRAS/TP53 Mutations in Pancreatic Juice.

We previously reported the usefulness of detecting aberrant methylation in tumor suppressive microRNAs (miRNAs) in bile and plasma to discriminate pancreaticobiliary cancers from benign pancreaticobiliary diseases. This study analyzed the methylation of miRNAs in pancreatic juice to identify those specific to pancreatic cancer (PC). Pancreatic juice was collected from 20 patients with PC, including eight with intraductal papillary mucinous carcinoma (IPMC), two with low grade-pancreatic intraepithelial neoplasia (LG-PanIN), 32 with LG-intraductal papillary mucinous neoplasm (IPMN), and seven with benign pancreatic lesions. Polymerase chain reaction amplification and sequencing were performed for three tumor suppressive miRNAs (miR-200a, 200b, and 1247), and their methylation rates were determined. Additionally, KRAS and TP53 mutations were analyzed. The methylation rate of miR-1247 was significantly higher in patients with PC than in those with LG-PanIN/IPMN. Furthermore, it was significantly higher in patients with IPMC than in those with LG-IPMN. KRAS and TP53 mutations were detected in seven (70%) and one (10%) of the patients with PC, respectively. Analyzing the methylation of miR-1247 in addition to KRAS and TP53 mutations in pancreatic juice may be useful to distinguish PC from PanIN/IPMN.

Analysis of composition of gut microbial community in a rat model of functional dyspepsia treated with Simo Tang.

To investigate composition of gut microbial community in a rat model of functional dyspepsia (FD) and to explore the interventional effects of Simo Tang (, SMT). A rat model of FD was established through the tail-clamping stimulation method. The rat model of FD was assessed by the state of rats, their weight, sucrose preference rate, and intestinal propulsion rate. The DNA was extracted from stool samples after treatment with SMT. Amplified polymerase chain reaction (PCR) products of the 16S rDNA were sequenced using NovaseQ6000 after construction of libraries. Composition of gut microbial community in the stool samples was determined and analyzed by cluster analysis, bioinformatic analysis, and analysis of α-diversity and β-diversity. The rat model of FD was successfully established using the tail-clamping stimulation method. The statistical results of cluster analysis of operational taxonomic units (OTUs) showed that the relative abundance of OTUs in the FD group was the lowest, while it was the highest in the normal (N) group. The composition of microbiome in the four groups was similar at phyla level. Compared with the FD group, the abundance of Firmicutes was downregulated, and the abundance of Proteobacteria and Bacteroidetes was upregulated in the Simo Tang (SMT) and high-dose Simo Tang (SMT.G) groups. The ratio of Bacteroidetes/ Firmicutes was also elevated. According to the analysis of α-diversity and β-diversity, the abundance of flora in FD rats was significantly reduced. The treatment using SMT appeared beneficial to improve the diversity of flora. SMT could improve the intestinal flora in FD rats. The results showed that FD rats had intestinal flora imbalance, and species diversity increased. The results suggested that SMT could regulate the disorders of intestinal flora caused by FD. SMT could restore gut homeostasis and maintain gut flora diversity by modulating the gut microbiota and its associated metabolites in rats, thereby treating gastrointestinal diseases.

A DNA sensor based on CbAgo effector protein and on a dual electrochemical signal amplification strategy for B19 parvovirus detection

Human parvovirus B19 is a prevalent childhood infectious virus that poses a great challenge to public health, so the detection of B19V is of great importance. In this study, a DNA sensor based on CbAgo, a Cas effector, and a dual electrochemical signal amplification strategy was developed by using a novel nanocomposite MnO2/CMK-3/g-C3N4/AgNPs for initial signal amplification, with CMK being an ordered mesoporous carbon nanomaterial. Single-walled carbon nanotubes (SWCNTs) were used as electrocatalytic probes for secondary signal amplification to detect B19 DNA. The detection process begins with polymerase chain reaction (PCR) amplification using the B19V infectious clone plasmid (pB19-M20) as a template and NS1-F/R as primers, followed by specific cleavage of B19 DNA based on the programmable cutting sites of CbAgo effector protein. This study enriches the application of Argonaute proteins in sensing and introduces a novel method to detect B19V. Under optimized conditions, the biosensor can detect B19 DNA in the range of 10−15–10−10 M, with a detection limit (LOD) of 0.2 fM. The results indicate that the developed DNA sensor holds promise for reliable and sensitive detection of B19 DNA in human serum.

Point-of-Care Diagnostics Using Self-heating Elements from Smart Food Packaging: Moving Towards Instrument-Free Nucleic Acid-Based Detection.

Compromising between accuracy and rapidity is an important issue in analytics and diagnostics, often preventing timely and appropriate reactions to disease. This issue is particularly critical for infectious diseases, where reliable and rapid diagnosis is crucial for effective treatment and easier containment, thereby reducing economic and societal impacts. Diagnostic technologies are vital in disease modeling, tracking, treatment decision making, and epidemic containment. At the point-of-care level in modern healthcare, accurate diagnostics, especially those involving genetic-level analysis and nucleic acid amplification techniques, are still needed. However, implementing these techniques in remote or non-laboratory settings poses challenges because of the need for trained personnel and specialized equipment, as all nucleic acid-based diagnostic techniques, such as polymerase chain reaction and isothermal nucleic acid amplification, require temperature cycling or elevated and stabilized temperatures. However, in smart food packaging, there are approved and commercially available methods that use temperature regulation to enable autonomous heat generation without external sources, such as chemical heaters with phase change materials. These approaches could be applied in diagnostics, facilitating point-of-care, electricity-free molecular diagnostics, especially with nucleic acid-based detection methods such as isothermal nucleic acid amplification. In this review, we explore the potential interplay between self-heating elements, isothermal nucleic acid amplification techniques, and phase change materials. This paves the way for the development of truly portable, electricity-free, point-of-care diagnostic tools, particularly advantageous for on-site detection in resource-limited remote settings and for home use.

Characterization, Genome Sequencing, and Development of a Rapid PCR Identification Primer for Fusarium oxysporum f. sp. crocus, a New forma specialis Causing Saffron Corm Rot.

Saffron corm rot (SCR), the most serious disease affecting saffron, has been confirmed to be caused by Fusarium oxysporum in previous studies. Compared to other fungal species, F. oxysporum exhibits host specialization, a special phenomenon associated with the secreted in xylem (SIX) genes. This study examined the pathogenicity specialization of F. oxysporum isolated from saffron corms with SCR disease. The results showed that this F. oxysporum strain was strongly pathogenic to saffron corms, causing SCR; weakly pathogenic to the corms of freesia, which is in the Iridaceae family along with saffron; and not pathogenic to watermelon, melon, and tomato. Other formae speciales of F. oxysporum were not pathogenic to saffron corms. This suggests that F. oxysporum saffron strains exhibit obvious pathogenicity specialization for Iridaceae spp. Subsequently, the F. oxysporum saffron strain (XHH35) genome was sequenced, and a comparative genomics study of XHH35 and three other formae speciales was conducted using OrthoVenn3. XHH35 contained 90 specific genes absent in the other three formae speciales. These genes are involved in certain key biological processes and molecular functions. Based on BLAST homology searching, the F. oxysporum saffron strain (XHH35) genome was predicted to contain seven SIX genes (SIX 4, SIX 6, SIX 7, SIX 10, SIX 11, SIX 12, and SIX 14) highly homologous to F. oxysporum f. sp. lycopersici, which was verified using polymerase chain reaction (PCR) amplification. The corresponding individual phylogenetic tree indicated that the F. oxysporum saffron strain (XHH35) showed a separate branch with different formae speciales. This study is the first-ever report of F. oxysporum f. sp. crocus, a new forma specialis. Based on the specificity of its SIX genes, the SIX 10 gene was selected to further establish a rapid identification technique for F. oxysporum f. sp. crocus, which will be useful in future research.

Development of triggerable peroxidase-tagged primers for colorimetric DNA detection applicable to various DNA polymerases

Abstract To meet the demand for more cost-effective rapid and sensitive nucleic acid detection methods to benefit various applications, we introduce a novel technique that uses a modified triggerable guanine quadruplex-based peroxidase (POD)-tagged primer. This technique allows sensitive colorimetric detection by activating an inactive POD tag during polymerase chain reaction amplification, which, importantly, functions independently of the type of DNA polymerase used.

Molecular and Clinical Spectrum of Hemoglobin D: An Experience at a Tertiary Care Diagnostic Center

Objective: To determine the clinical and molecular pattern of Hemoglobin D in patients presenting at tertiary care hospital. Study Design: Cross-sectional study. Place and Duration of study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Jul 2020 to Mar 2021. Methodology: An automated cell analyzer was used to determine the whole blood count and red cell indices. D-10 BIORAD HPLC was used for quantitative assessment of Hb A, Hb F, Hb D and Hb A2. Where D-10 BIORAD HPLC showed an abnormal Hb D, capillary electrophoresis was performed using Sebia Capillary 2 Flex Piercing. Chelex DNA Extraction method was used. Molecular analyses for the confirmation of homozygosity and heterozygosity were done using Polymerase Chain Reaction (PCR) amplification. The clinical symptoms of the patients such as anemia, jaundice, pallor, weakness, hepatomegaly and splenomegaly were also noted Results: Among 90 patients, Hemoglobin D trait was found in 78(80.4%) of the patients out of which HbD-Punjab was seen in 57(73%) and HbD-Iran was found in 21(27%). Compound Heterozygotes (HbD/β) was found in 17(17.5%) and Compound Homozygous HbD/D was found in 02 (2.1%). All HbD-Punjab and HbD-Iran traits patients were asymptomatic, and few clinical symptoms were found in Compound Heterozygotes (HbD/β) and Homozygous HbD/D. Conclusion: Collective clinical history records, full blood count and electrophoresis and molecular results allow for the conclusive detection of the variant of Hemoglobin D. More multicenter, organized and detailed studies with larger sample size are required to draw concrete conclusions.

Effects of energy-gathered and energy-divergent ultrasound on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch.

The identification of adulterated sweet potato vermicelli faces significant challenges, seriously hindering the development of the vermicelli industry. Herein, we investigate effects of energy-gathered ultrasound (EGU) and energy-divergent ultrasound (EDU) (30, 40 and 50 W L-1) on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch, thereby exploring their potential in adulteration of sweet potato vermicelli. EGU-assisted modified cetyltrimethylammonium bromide (CTAB) protocol with β-mercaptoethanol significantly improved DNA extraction from sweet potato vermicelli (223.7-249.2 ng μL-1) and its starch (133.4-186.4 ng μL-1), followed by EDU-assisted DNA extraction from sweet potato vermicelli (115.1-209.3 ng μL-1) and its starch (33.4-61.0 ng μL-1). Both EGU and EDU treatments resulted in the destruction of microstructure and crystalline structure, as well as changes in pasting and thermal properties of sweet potato vermicelli and its starch. Real-time polymerase chain reaction (PCR) amplification results revealed that EGU and EDU enhanced the efficiency of DNA amplification, and EDU showed smaller cycle threshold (Ct) values than EGU. In addition, EDU-assisted CTAB protocol combined with real-time PCR could detect levels of less than 1% of cassava and maize starches in sweet potato vermicelli. EDU-assisted CTAB protocol combined with real-time PCR shows promise as a sensitive and reliable analytical tool for vermicelli adulteration. © 2024 Society of Chemical Industry.

Virulence and molecular epidemiological analysis of three human blood-borne Streptococcus suis

ObjectiveTo understand the virulence genes and molecular epidemiological characteristics of human-infected strains of Streptococcus suis in Rui'an, Zhejiang Province, from 2021 to 2022, and to provide a scientific basis for diagnosis, treatment, prevention, and control. MethodsThree blood-borne strains of Streptococcus suis were analysed by morphological observation, identification, and drug sensitivity tests. We performed polymerase chain reaction (PCR) amplification of their main seven virulence factors and housekeeping genes. This was followed by virulence analysis and multilocus sequence typing. We analysed their relationships with local pathogens from previous years. ResultsThree Streptococcus suis strains were isolated from the blood samples of three patients. From these, the virulence genotypes demonstrated that the two strains were orf2+ and ef+/orf2+/sly+, respectively. The Multilocus sequence typing (MLST) typing results demonstrated that the two strains were ST25 and ST7, respectively. ConclusionThe first isolation of ST25 Streptococcus suis in Rui'an was presumed to have a close affinity with the endemic strain in North America. The other strain was an ST7 clone, consistent with the endemic strain in Sichuan, and which may have originated from Sichuan. Virulence genotype analysis demonstrated that different virulence genes of the pathogens resulted in different clinical manifestations.

Uncommon Invasive Penicillium Species Infection in a Patient with Advanced HIV: A Rare Case Report

Penicillium species are ubiquitous worldwide and constitute one of the largest fungal genera. Typically benign, Penicillium (P.) non-marneffei species can become a serious threat in immunocompromised hosts with the potential for high mortality. We present a rare care of disseminated P. non-marneffei infection in a Honduran patient with advanced HIV, initially manifesting as nonspecific symptoms. After a thorough and unrevealing workup, an inguinal lymph node biopsy resulted in positive fungal staining of tissue. However, expanded polymerase chain reaction (PCR) amplification of fungal 28S rDNA was necessary to confirm the diagnosis. Here we describe the first reported case of disseminated infection in a patient with HIV/AIDS presenting with lymphadenitis and propose treatment recommendations as no standards have been developed yet.

Sweet enhancers of polymerase chain reaction

Although faster and powerful, polymerase chain reaction (PCR) often failed to amplify targets efficiently. Numerous PCR enhancers have been used to increase the amplification efficiency of difficult DNA targets. However, there is no systematic comparison of their effects in normal and difficult PCR conditions. In this paper, we have selected nine different PCR enhancers that can promote the PCR amplification efficiency. We have compared their effect in Taq DNA polymerase thermostability, inhibitor resistance, and amplification of various DNA targets. Although the PCR enhancers more or less reduced the amplification efficiency of DNA fragments with moderate GC-content, they were able to improve the amplification efficiency and specificity of GC-rich fragments. Betaine outperformed the other enhancers in amplification of GC-rich DNA fragments, thermostabilizing Taq DNA polymerase, and inhibitor tolerance. Sucrose and trehalose showed similar effect in thermostabilizing Taq DNA polymerase and inhibitor tolerance, while they showed mildest inhibitory effect on normal PCR. For GC-rich region-containing long DNA fragment amplification, 1 M betaine, 0.5 M betaine + 0.2 M sucrose, or 1 M betaine + 0.1 M sucrose can be used to effectively promote the amplification, while keep their negative effect in amplification of normal fragment to a minimal level.

Morphological and Molecular Characterization of Trichotylenchus dispersus (Nematoda: Dolichodoridae), a Newly Recorded Plant-Parasitic Nematode in the Rhizosphere Soil of Tomato Plants in Thailand

Trichotylenchus sp. is a migratory ectoparasitic nematode (PPN) that causes hypoplasia disease symptoms in economic plants, such as maize and banana. In this study, soil samples were collected from 6 locations in a tomato field in Khon Kaen Province, Thailand, for the purpose of nematode extraction using the Cobb’s sieving and flotation-centrifugation techniques. Morphological and molecular characterization of the collected PPN identified it as Trichotylenchus sp., a PPN not previously found in Thai tomato fields. Morphologically, the nematode has a C-shaped body, a rounded lip region, robust stylet and dorsal gland orifice located 2.0 ± 0.5 µm from the base of basal knobs. The tail is conically shaped, and the cuticle exhibits annulations with 3 incisures in the lateral field. The morphological characteristics observed closely matched Trichotylenchus sp. To confirm the identity, diagnosis was done using Polymerase Chain Reaction (PCR) molecular technique. Nematode DNA amplification was conducted with different target genes using primer sets AB28/TW81 for ITS1-5.8S-ITS2 and D2A/D3B for D2-D3 region of the 28S rRNA. The PCR amplification showed DNA fragments of approximately 950 and 800 bp, respectively. The sequences obtained were compared with those in the NCBI GenBank, which showed a 98.0 - 99.4 % identity match with Trichotylenchus dispersus specimens previously documented in China. In addition, the phylogenetic trees reiterated a nematode grouping with T. dispersus, 100 % bootstrap value. To the best of our knowledge, this is the first report of the presence of T. dispersus in the soils of tomato field in Thailand. This nematode is likely to become a major factor limiting tomato production yields if not properly managed. HIGHLIGHTS Six plant-parasitic nematodes (PPNs) were found to be associated with the tomato gown in field. A newly recorded PPN in the genus, Trichotylenchus, found in Thailand, has been documented in this study. Morphological and molecular characterization identified PPN as Trichotylenchus disperses. This is the first report on incidence of dispersus in a tomato field in Thailand. GRAPHICAL ABSTRACT

Streamlined boiling lysis DNA extraction for Gram-positive aquaculture pathogen Streptococcus agalactiae.

Accurate genetic analysis is essential for the detection of pathogens as it necessitates suitable DNA extraction methods tailored to specific microorganisms such as Gram-positive bacteria. This study examined several commercial and simplified DNA extraction methods for their suitability in isothermal downstream applications. Extracted DNA was assessed using spectrophotometry, electrophoresis, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) while its stability was inspected after five months of storage. The findings revealed variations in DNA yield, purity and integrity among the extraction methods. While extraction kits demonstrated high yield and purity, the in-house extraction techniques showed incoherent correlation between yield and purity, yet showed promise for a streamlined extraction process. The DNA acquired from all methods yielded positive amplification in PCR and LAMP. DNA extracted by kits exhibits prolonged stability than those obtained via boiling lysis. Both methods offer distinct advantages, with commercial kits providing longer stability and high-quality DNA while boiling lysis stands out for its simplicity, with shorter handling and processing periods. This study emphasizes selecting ideal extraction methods for Streptococcus agalactiae, in the prospect of aquaculture settings. In particular, successful LAMP reaction suggests that boiled extracts are feasible enough for detection, and suited for point-of-care (POC) testing where prompt detection of aquatic pathogens is often critical. Ultimately, the choice of method should be contemplated on a case-by-case basis such as the study goals, intended settings, and type of samples.

Repeat-mediated recombination results in Complex DNA structure of the mitochondrial genome of Trachelospermum jasminoides

BackgroundTrachelospermum jasminoides has medicinal and ornamental value and is widely distributed in China. Although the chloroplast genome has been documented, the mitochondrial genome has not yet been studied.ResultsThe mitochondrial genome of T. jasminoides was assembled and functionally annotated using Illumina and nanopore reads. The mitochondrial genome comprises a master circular molecular structure of 605,764 bp and encodes 65 genes: 39 protein-coding genes, 23 transfer RNA (tRNA) genes and 3 ribosomal RNA genes. In addition to the single circular conformation, we found many alternative conformations of the T. jasminoides mitochondrial genome mediated by 42 repetitive sequences. Six repetitive sequences (DRS01–DRS06) were supported by nanopore long reads, polymerase chain reaction (PCR) amplifications, and Sanger sequencing of the PCR products. Eleven homologous fragments were identified by comparing the mitochondrial and chloroplast genome sequences, including three complete tRNA genes. Moreover, 531 edited RNA sites were identified in the protein-coding sequences based on RNA sequencing data, with nad4 having the highest number of sites (54).ConclusionTo our knowledge, this is the first description of the mitochondrial genome of T. jasminoides. Our results demonstrate the existence of multiple conformations. These findings lay a foundation for understanding the genetics and evolutionary dynamics of Apocynaceae.

Streamlining RNA Aptamer Selection via Unique Molecular Identifiers and High-Throughput Sequencing.

Aptamers are valuable tools for applications such as cell imaging, drug delivery, and therapeutics. RNA aptamers, in particular, exhibit complex structural diversity and flexibility, affording higher affinity and specificity, broader target recognition, and easier chemical modification compared with DNA aptamers. However, traditional selection methods for RNA aptamers are time-consuming and involve numerous rounds of screening, thus limiting their widespread application. To overcome this challenge, we propose an efficient truncated selection approach termed ID-SELEX. This method incorporates a molecular identification marker whereby each template is labeled with a unique molecular identifier, or UMI. Such incorporation helps mitigate biases introduced by multiple polymerase chain reaction (PCR) amplification during high-throughput sequencing, ensuring accurate identification of aptamer candidates. Utilizing ID-SELEX, we successfully identified a panel of high-quality aptamers targeting the human colon cancer cell line HCT-8 in just 2 rounds of selection. Furthermore, we demonstrated the versatility of this strategy by selecting 6 RNA aptamers targeting mouse myoblast cell line C2C12 with only one round of selection. In summary, RNA aptamer selection based on ID-SELEX utilizes high-throughput sequencing and UMI labeling to enable the rapid screening of RNA aptamers across human and murine cell lines. As such, ID-SELEX has the potential to facilitate RNA aptamer discovery, providing a novel molecular tool for biomedical research, clinical applications, and precision medicine.

Isolation and pathogenicity of a fowl adenovirus 8b (FAdV-8b) strain in Cherry Valley ducks

ABSTRACT Inclusion body hepatitis (IBH) is an economically important viral disease primarily affecting the poultry industry. In this study, we isolated a strain of FAdV-8b (strain SDYT) from naturally infected ducks and the hexon and fiber gene sequences were analysed by polymerase chain reaction (PCR) amplification. In order to study the pathogenicity of FAdV-8b in Cherry Valley ducks, we inoculated 10- and 20-day-old ducks with 0.3 ml of FAdV-4 virus (TCID50 of 105.5/0.1 ml) either orally or intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines, viral load and antibody levels were observed and recorded within 15 days after infection. Pathomorphological investigations revealed that ducks in the experimental group exhibited hepatitis. Histopathology showed multiple organ damage, including serious liver and kidney lesions. Furthermore, elevated levels of inflammatory cytokines and antibodies were noticed, due to the infection and innate immune response. At a later stage of infection, immunosuppression occurred, resulting in decreased levels of cytokines. Determination of viral load showed that the virus was present in several organs, with the highest viral DNA load found in the liver, followed by the kidney. Compared to birds infected orally, the intramuscular group exhibited the highest viral load. In summary, this study increases our understanding of the pathogenicity of FAdV-8b in ducks and establishes a model that will inform antiviral drug testing and vaccine evaluation for IBH, thereby preventing and reducing the spread of IBH in the poultry industry. RESEARCH HIGHLIGHTS A strain (SDYT) of fowl adenovirus 8b (FAdV-8b) was successfully isolated from ducks. Cherry Valley ducks were successfully infected with FAdV-8b. Different routes of infection can lead to duck mortality, more pronounced when birds are injected intramuscularly. FAdV-8b (SDYT) was distributed in various tissues and organs of ducks, causing different degrees of lesions.

Clinical significance of Spinal Muscular Atrophy carrier detection in Guangdong Province, China: Insights from quantitative polymerase chain reaction and multiplex ligation-dependent probe amplification analysis

ObjectiveThis study aimed to identify carriers of Spinal Muscular Atrophy (SMA) among 10,630 pregnant women in Guangdong Province and provide prenatal diagnoses for high-risk fetuses from carrier couples. The goal was to prevent the birth of children affected by SMA. We evaluated the effectiveness of quantitative PCR (qPCR) and multiplex ligation-dependent probe amplification (MLPA) in detecting deletions in the SMN1 gene, with MLPA as the reference standard. MethodsFluorescent qPCR was used for initial SMA carrier screening, followed by confirmatory testing with MLPA for all detected carriers. ResultsOf the 10,630 women screened, 219 were identified as carriers (2.06 % detection rate). This included 17 cases of heterozygous deletion of exon 7 (E7), 145 cases with deletions of both E7 and exon 8 (E8), and 57 cases of E8 deletion alone. The carrier rate for E7 heterozygous deletion was established at 1.5 %. Prenatal diagnosis for seven carrier couples revealed five fetuses as carriers and one affected by SMA. The diagnostic concordance between qPCR and MLPA was 100 %. ConclusionThe combined use of qPCR and MLPA is vital in identifying SMA carriers, allowing for early diagnosis and informed reproductive decisions. The high sensitivity and specificity of qPCR, matching MLPA, demonstrate its value in clinical settings for SMA screening and prenatal diagnosis. Our findings emphasize the critical importance of selecting precise diagnostic methods to enhance clinical outcomes in genetic screening programs.

A-272 Evaluation of a Real-Time Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Respiratory Pathogens in Nasopharyngeal Specimens

Abstract Background Respiratory infections present a significant challenge due to the diverse range of pathogens involved and the possibility of concurrent infections, emphasizing the importance of rapid and accurate detection for effective treatment. Advancements in molecular methods have provided cost-effective and reliable solutions for the identification of respiratory pathogens. In this study, we aimed to assess performance characteristics of the Seegene Allplex™ 2019-nCoV Assay and Seegene Novaplex™ Respiratory Panel Assays for simultaneous amplification and detection of 22 respiratory pathogens using real-time multiplex polymerase chain reaction (PCR). Methods Nucleic acid extraction was performed using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, MA) on the CyBio® FeliX liquid handler (Analytik Jena, Germany). PCR amplification and detection were performed using the Seegene Novaplex™ Respiratory Panels 1A, 2, 3, PneumoBacter Assay, and Allplex™ 2019-nCoV Assay (Seegene Inc, South Korea) on the CFX96™ System (Bio-Rad Laboratories, CA). The assays utilize Seegene MuDT™ technology that allows for the detection of multiple targets in a single channel without the need for melt curve analysis. For method validation, precision studies were performed within-run and over 4 days. The limit of detection was verified by using plasmids from Seegene containing the target sequence for each pathogen at known concentrations. Method comparison studies were carried out by extracting and analyzing a range of 41 to 79 split samples for each pathogen against the BioFire® Respiratory 2.1 Panel (bioMérieux, France), cobas® Influenza A/B & RSV Assay on the cobas® liat system (Roche Diagnostics, IN), and Lyra® SARS-CoV-2 Assay (Quidel, CA) on the CFX96™ system. Additional accuracy studies were performed by spiking negative patient samples with standards from ZeptoMetrix® (Antylia Scientific, IL) containing known concentrations of the targeted respiratory pathogens. Results Precision studies demonstrated consistent and reproducible results for all pathogens. The limit of detection was verified at 1000 copies/reaction for human parainfluenza virus 4 and human metapneumovirus, and at 100 copies/reaction for all other pathogens. Results of the spike-and-recovery studies were consistent with expected results for all targets. Patient correlation studies showed 100% agreement with expected pathogen identification for 19 pathogens, and greater than 97.9% agreement for influenza B virus, respiratory syncytial virus, human parainfluenza virus 4, and human coronavirus 229E. Conclusions The Seegene Allplex™ 2019-nCoV Assay and Novaplex™ Respiratory Panel Assays offer a rapid and reliable method for detecting respiratory pathogens in nasopharyngeal specimens. The capability of the assays to detect multiple targets in a single well enhances efficiency, cost-effectiveness, and turnaround time.

B-236 New real-time PCR test for APOE genotyping in patients with Alzheimer’s disease before anti-amyloid therapy

Abstract Background In 2023, the US Food and Drug Administration approved LEQEMBI, a monoclonal antibody therapy targeting beta-amyloid plaques, for patients with early-stage Alzheimer’s disease (AD). However, anti-amyloid drug trials demonstrated an elevated risk for amyloid-related imaging abnormalities with cerebral edema (ARIA-E) in carriers of the AD risk allele apolipoprotein E epsilon 4 (APOE-ɛ4), especially in ɛ4/ɛ4 homozygous individuals [1-3]. Here, we report on the evaluation of a new real-time polymerase chain reaction (PCR) test for APOE genotyping. Methods The EURORealTime APOE (EUROIMMUN), which enables simultaneous testing for all six APOE genotypes (ɛ2/ɛ2, ɛ2/ɛ3, ɛ2/ɛ4, ɛ3/ɛ3, ɛ3/ɛ4, and ɛ4/ɛ4) by real-time PCR, was performed using genomic DNA (gDNA) isolated from EDTA blood samples. The PCR amplification results were evaluated using the EURORealTime Analysis Software (EUROIMMUN). Samples from healthy blood donors (HBD) were used to assess intra-assay, inter-assay and between-lot precision (n=6) as well as analytical sensitivity (n=21), with each APOE genotype represented by at least one sample. Assay accuracy was analyzed by comparing genotype assignments to the results of the CE-IVD-marked EUROArray APOE (EUROIMMUN) and bidirectional Sanger sequencing using 110 samples (100 AD, 10 HBD). APOE genotype frequencies were compared between patients (100 AD) and controls (250 HBD). Results Precision testing of the EURORealTime APOE resulted in 100% correct genotype calls. Assessment of the analytical sensitivity revealed valid and correct genotype assignment in all 21 samples, confirming 1 ng/µl as the recommended minimum gDNA concentration. The analytical accuracy amounted to 100% based on agreement with the two reference methods in 110/110 cases. Comparison of the APOE genotype distribution showed a higher proportion of ɛ4 allele carriers (i.e., ɛ2/ɛ4, ɛ3/ɛ4, and ɛ4/ɛ4) in AD patients than in HBD (50.0% vs. 25.2%). The prevalence of the ɛ4/ɛ4 genotype in these cohorts was 25.0% for AD patients compared with 2.8% for HBD. Conclusions The well-established high prevalence of the APOE-ɛ4/ɛ4 genotype in AD patients, together with the increased risk of ARIA-E in these homozygous carriers, substantiates the relevance of APOE genotyping prior to anti-amyloid treatment. The EURORealTime APOE test provides sensitive and reliable determination of APOE alleles, thus presenting a suitable tool to improve risk stratification for potential LEQEMBI recipients.