bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-224 Comparison of an Unlabeled Probe High Resolution Melting Analysis (HRMA) Assay for Factor V Leiden 1691 G &gt; A Mutation to a TaqMan Hydrolysis Assay
Authors: Han H H , Lewis S , Hoang A , Badri K 
Published: September 2023

Abstract

Background
The clinically significant Factor V Leiden (FVL) mutation (1691 G &gt; A) causes replacement of Arg with Gln, preventing activated protein C from inactivating Factor V leading to a lengthened clotting process. Individuals with the Factor II and Factor V Leiden mutations have an increased risk for venous thrombosis. The aim of this study is to compare an unlabeled probe High Resolution Melting Analysis (HRMA) assay for Factor V Leiden mutation to a TaqMan hydrolysis assay. High Resolution Melting Analysis (HRMA) is a post-PCR, homogenous, closed tube system for the detection of sequence variants. Post-PCR, the amplicons are heated gradually until the melting temperature is reached and the fluorescent dye unbinds from the amplicon and exhibits low fluorescence. A melt-curve analysis is generated that is characteristic of a particular sequence variant, therefore HRMA allows for comparison of one base changes in genetic sequences based on their differences in melting rate.

Methods
HRMA Assay: Blood samples were collected in EDTA tubes and DNA extracted using the Roche MagnaPure. DNA content was determined to supply 66 ng of genomic DNA to each reaction mixture. Factor Vc.1601 G &gt; A Novallele Genotyping Assay by Canon Biosciences was used. The following primer and probe sequences were used: forward primer sequence: 5′-CCCATTATTTAGCCAGGAGA-3′, reverse primer sequence: 5′-GCCTCTGGGCTAATAGGACT-3′, unlabeled probe sequence: 5′-TTCAAGGACAAAATACCTGTATTCCTCGCCT/3AmM/3′. PCR was performed in Roche Light Cycler 480 with 11 uL volumes per reaction of 66.6 ng of DNA. Forward primer, reverse primer and probe concentration was 100 uM and a 5× volume excess amount of forward primer and probe was used. A 151 bp amplicon was generated. Three control references obtained from Coriell Institute were used including wild type (WT), mutant (MUT) and heterozygote (HET). The HRMA FVL assay was performed on a total of seventeen samples. The TaqMan hydrolysis FVL assay uses the following DNA primers and probe: forward primer: GCCTCTGGGCTAATAGGACTACTTC; reverse primer: TTTCTGAAAGGTTACTTCAAGGACAA; WT FVL probe: HEX-ACC TGT ATT CCT CGC CT-BHQ-2; MUT FVL probe: FAM-ACC TGT ATT CCT TGC CT-BHQ-2. Reactions were performed in a 20 ul volume with 50 ng of DNA per reaction. The same sets of reference DNA and 17 samples of DNA as the high-resolution melting analysis were used.

Results
For FVL, the Tm was 65.58 °C and 69.75 °C respectively for the WT and MUT. Tm for all 17 samples was 69.75 °C respectively indicating all 17 samples were WT FVL. This TaqMan FVL assay confirmed the FVL genotype of the 17 samples and reference samples.

Conclusion
Comparing the results of the unlabeled probe, HRMA FVL assay with a real-time TaqMan probe endpoint genotyping assay resulted in 100% sensitivity and 100% specificity for both assays.
B-232 Versatile, Extraction-Free Nucleic Acid Sample Preparation in 15 Seconds Via Hyperbaric Heating
Authors: Hambalek J A , Martin R L , Alvarado E , Halle P W , Davis R E , Janwari H , Kido H , Peytavi R 
Published: September 2023

Abstract
Nucleic acid extraction-based PCR sample prep is currently limited by long processing times, liquid transfers, kit cost, and lab storage requirements. Extraction-free approaches enable streamlined sample prep, but lack sensitivity compared to extraction-based methods. We studied several sample types to demonstrate the effectiveness of a novel 15-second Hyperbaric Heating (HBH) process to prepare nucleic acids for PCR. HBH exposes the sample to a short duration, high temperature environment to effectively lyse organisms and denature PCR inhibitors such as nucleases in seconds. Samples are introduced and sealed in a pressure-tight metallic vessel, which is then heated in seconds using magnetic induction. Within the sealed metallic vessel, the vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C to be achieved. Chelating agents and other proteins in HBH also protect the target nucleic acids from fragmentation. Post HBH, the sample is ready for direct PCR amplification without further dilution or extraction. Efficacy of HBH for efficient PCR amplification was tested against commercial kits. HBH was applied to viral, bacterial, eukaryotic, and fungal organisms. For SARS-CoV-2, HBH detected down to 250 viral copies/mL. Additionally, HBH outperformed commercial kits for PCR sample prep of Candida albicans and Bacillus subtilis. HBH is a generalizable sample prep technology with sensitivity comparable to that of current extraction kits. We have applied HBH to a multitude of organisms and have demonstrated that HBH can be employed as a rapid and comprehensive nucleic acid prep approach for applications that involve PCR amplification post sample prep. Future integration of HBH in devices suitable for point of care molecular testing are underway. We also envision HBH to be a useful, generalizable sample prep method for the molecular lab in applications where cost, simplicity, and speed are essential for preparing nucleic acids for amplification.
B-234 The Quality in NGS: Do You Know How Much We Lost?
Authors: Chao T , Chen P , Huang W 
Published: September 2023

Abstract

Background
Next generation sequencing (NGS) is a new high throughput approach for genomic sequencing and variations detection. The consumption of NGS is numerous, such as times, money and human resource, therefore, some hospitals provided the service but delivered the specimen to other laboratories. Hence, we aimed to establish a procedure to monitor the quality and integrity of report.

Method
Here we used one clinical case to elucidate the whole process of raw sequence data analysis by free resource, a website usegalaxy.org and the IGV visualization software, with flowchart and quality control (QC) items. A delivered case will be an example to show how to monitor the QC.

Results
The flow chart showed the whole steps of analysis of NGS, including evaluation genetic variants in the report from individual laboratory. We processed the FASTQ file and analyzed the quality with three parameters. An overall picture of bases showed the phred score (QV) less than 28. Distribution of sequence lengths over all sequences highlighted a prominent peak at 110–119 bp. The mean QV of reads was around 26 with low quality (QV less than 20) in some reads. We then filtered the FASTQ file and the reads with length less than 45 bp or greater than 150 bp and QV less than 20 were filtered out. The data of the BAM file mapped to the designed panel were output. There were 8252 genome regions designed over 23 chromosomes (chr). A total number of reads was 1 085 653 with the average 131.6 reads (range: 0–31034; quartiles, IQ25: 1; IQ50: 27; IQ75: 152). 7746 of designed genome regions (93.9%) had reads less than 500 (quartiles, IQ25: 0; IQ50: 20; IQ75: 118). We further used IGV to check one of mutations which showed KRAS G12V in the report. KRAS G12V (chr.12: 25245350) was read 25 times with the variant allele frequency (C to A) of 24%.

Conclusion
We showed how to monitor the NGS quality by free resource. It is important to ensure high-quality data after we received the individual laboratory reports. That may help increase the quality of patient care and clinical service.
B-239 Long-read Nanopore Sequencing is a Practicable Method of Targeted or Untargeted Microbial Contamination Detection
Authors: Suhandynata R , Maheu S , Safiari S , Bevins N 
Published: September 2023

Abstract

Background
Sequencing-based methods for microbe detection are poised to replace both in vivo and PCR-based detection methods due to advantages in speed, sensitivity, specificity, and multiplexing. Here we describe pilot experiments to assess the practicability of nanopore sequencing for microbe detection.

Methods
Quantitative genomic Human Adenovirus 5 or Mycoplasma hominis DNA was spiked into remnant human serum and nucleic acids were extracted. 16S rRNA PCR amplification and barcoding was performed for Mycoplasma hominis identification. Sequencing was performed with a R9.4.1 flowcell on the MinION sequencer. Basecalling and genome alignment was performed with Guppy v.6.4.2.

Results
The performance of untargeted sequencing was evaluated by spiking quantitative Adenovirus DNA at 104 genome copies per microliter. Illustrative data from a single run is presented. Replicates yielded similar results. 1 520 991 reads between 31 bases and 970 kilobases were generated. 162 reads between 700 bases and 11.3 kilobases aligned to the Adenovirus genome (Image 1).
To evaluate the performance of PCR amplified sequencing, we spiked Mycoplasma genomic DNA into serum or water. The entire 16S rRNA gene (1.5 kb) was PCR amplified and genomic DNA was spiked in at 104, 102, 10, and 0 (control) copies per microliter. A 10% signal: noise ratio cutoff yielded 100% specificity between spiked samples and negative controls.

Conclusion
Short read sequencing techniques are adequate for microbial detection but are capital intensive and prone to false positive results. We demonstrate that nanopore sequencing can generate long reads (&gt;1.5 kb) with high-confidence microbe genome alignment. Our methods were sensitive to 104 and 10 copies per microliter utilizing untargeted and targeted methods, respectively. Further method optimization and validation will need to be performed, but these results demonstrate that nanopore sequencing is a practicable approach for microbe detection.
B-243 Utilizing Lyophilized LAMP Reagents in Rapid Molecular Assays
Authors: Alpaslan E , Zhang Y , Tanner N A , Patton G C 
Published: September 2023

Abstract

Background
Loop Mediated Isothermal Amplification (LAMP) has become a widely used method for detection of target nucleic acids (DNA and RNA) as it offers a robust and simple alternative to PCR. LAMP is particularly well suited to point of care (POC) applications such as COVID-19 diagnostics because only a single incubation temperature is required for nucleic acid amplification and the technology is compatible with simple detection strategies, including colorimetric or lateral flow readouts. To further increase the utility of LAMP in POC molecular diagnostic workflows, elimination of cold chain requirements for reagent shipment and storage is desired. In this study, we investigate single and multiplex detection of several viral infectious diseases using lyophilized RT-LAMP reagents.

Methods
Assays targeting SARS-CoV-2, FluA, FluB and a host control nucleic acid were evaluated using various detection methods with lyophilized RT-LAMP reagents. Compatibility was established with endpoint LAMP detection strategies, such as colorimetric dyes, and real time multiplex LAMP detection, which is increasingly finding utility in POC testing for common respiratory infections that present with similar symptoms.

Results
Assays employing lyophilized RT-LAMP reagents maintained robust detection of viral respiratory targets within 30 min at 65°C using various detection strategies. Colorimetric dyes such as eriochrome back T and calcein resulted in successful visual readout upon amplification while, one prominent colorimetric dye, hydroxy naphthol blue, was found to be incompatible with the lyophilized reagents used in the study. Additionally, simple hybridization probes permitted simultaneous detection of SARS-CoV-2, FluA and FluB in a single reaction.

Conclusion
LAMP is a robust, simple, isothermal amplification strategy that continues to enable testing beyond traditional laboratory settings. Lyophilized reagents extend the utility of LAMP in POC settings regardless of the detection strategy and will be important factors in the extension of this powerful molecular diagnostic tool to decentralized, field, and at-home testing applications.
B-253 Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing
Authors: Yang C , Wang M , Chang S , Huang C , Chang P , Chueh T 
Published: September 2023

Abstract

Background
High-resolution human leukocyte antigen (HLA) typing is a crucial process in transplant matching. Sequencing-based typing (SBT) has been the gold standard method used in HLA-based clinical applications. Recently, next-generation sequencing (NGS) has been adopted in clinical laboratories for HLA typing. NGS HLA typing assays cover whole or near-whole target HLA genes, allowing the detection of genetic variants outside of key exons. Multiplex amplicon-based NGS is an efficient high-resolution HLA typing method with minimal ambiguity and a high throughput. Here, we report the performance and validation of NGS for HLA typing and compare the results obtained using NGS with those of SBT to identify any discrepancies between the 2 methods.

Methods
A total of 237 samples from 2019 to July 2022 were collected from Chang Gung Memorial Hospital, including 42 samples previously subjected to SBT sequencing for 4-digit HLA analysis. NGS was performed for 5 whole genes (HLA-A/B/C/DQA1/DPA1) and 3 near-whole genes (HLA-DRB1/DQB1/DPB1; excluding exon 1 and part of intron 1) by using the AllType NGS kit (One Lambda), Illumina MiSeq platform, and Type Steam Visual NGS analysis software (One Lambda).

Results
Our validation process comprised 34 NGS runs and 237 samples. In total, 42 samples were analyzed with an overall accuracy and precision of 100% and 100% reproducibility. The overall ambiguity rate was 0% in HLA-A/B/C compared with 56.9% for SBT. Two discrepant alleles of HLA-A and DRB1 were noted in NGS and Sanger sequencing. In accordance with additional SBT exon analyses, one of the results was corrected according to the NGS results.

Conclusion
NGS HLA typing is a high-throughput approach with high accuracy and precision and a low ambiguity and is promising for use in clinical HLA laboratories.
B-256 Genotype Distribution of the Hepatitis C Virus: 10 Years Data of a Clinical Laboratory in Brazil
Authors: Nurmberger J M , de Carvalho G C , Perez B B , Borges J G , Ramadan D R , Tufik S 
Published: September 2023

Abstract

Background
Hepatitis C virus (HCV) infection is a significant health problem worldwide and is a major cause of severe liver disease including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is divided into six genotypes with several sub-genotypes depending on the geographic region, some genotypes are endemic. The purpose of this study was to describe the frequency of the HCV genotype (G) profile by gender and age using a database of a large Brazilian clinical laboratory.

Methods
This was a retrospective analysis from January 2012 to December 2022 based on molecular data of 3.566 isolated from clinical samples. The automated extraction and purification of nucleic acids were conducted from EDTA plasma samples of individuals suspected of HCV infection. Then, nucleic acid amplification was performed using oligonucleotide probes labeled with fluorescent dyes specific for the qualitative identification of HCV genotypes 1 to 6 and subtypes A and B of genotype 1.

Results
A total of 3.566 HCV genotype cases were analyzed during the period. The genotype distribution was (2.653/74.4%) G1 (48.1% G1A and 46.4% G1B), (733/ 20.5%) G3, (133/3.7%) G2, (35/0.9%) G4, (11/0.3%) G5 and (1/0.02%) G6. Twelve patients had multiple genotypes over the last ten years. At least one individual changed the genotype over a 3-year evaluation period, and 11 (0.30%) changed it twice. The genotypes G1 and G3 were predominant in all years analyzed. The prevalence of G1 and G3 was 75% and 19.6% among females and 74% and 21.2% among male patients, respectively. The analysis was conducted on individuals aged 6 to 99 years, with an overall average age of 52.6±13 years. The average age among men was 51.1 ± 12 years and among women, it was 54.7 ± 13 years. According to the age groups evaluated, genotype 1 was the most frequent, ranging from 69.3% (21 to 30 years old) to 75.8% (over 60 years old). The main subtype in the over 60 years old group was 1B. The highest frequency of genotype 2 was observed in the group over 60 years old (6.1%). The patient's symptoms, clinical history, or further diagnostic procedures were not accessible.

Conclusion
The study found that genotypes 1 and 3 were the most prevalent in Brazil over the ten-year period, with genotype 1 being the most frequent among all age groups. Genotype 1B was most prevalent in individuals above 60 years old. The change of genotype per individual was less than 1%, but further attention is required for potential virus tropism.
B-268 Validation of a New Real-Time PCR Test for the Detection of Mpox Virus on the BD MAXTM System
Authors: Marín-Royo G , Palacios-Pérez C , Aznar-García A , Llobet-Sesé L , García-Manrique B 
Published: September 2023

Abstract

Background
Enabling labs to rapidly respond to unexpected healthcare challenges is the kind of situations for which the BD MAX™ open system reagent suite is designed. Therefore, after the monkeypox outbreak in non-endemic countries on May 2022, development of a molecular assay for monkeypox detection on the BD MAXTM System was initiated.

Methods
VIASURE Monkeypox virus Real Time PCR Reagents for BD MAX™ System was designed for the qualitative detection of DNA from monkeypox virus in human skin lesion swabs in VTM/UTM obtained from individuals suspected of monkeypox infection.
The Limit of Detection (LoD) was determined by testing a 3-fold dilution series across 5 concentrations. The lowest concentration with 100% detection was tested with an additional 20 replicates. The preliminary LoD was confirmed with a ≥95% detection with 20 replicates. Then, a fully contrived clinical evaluation was performed. Each contrived clinical specimen was prepared using a unique natural clinical specimen matrix (lesion swabs). Twenty (20) of the clinical specimens were prepared at 1-2X LoD, ten (10) were prepared at 3-5X LoD, and thirty (30) were tested as negative samples.

Results
The lowest detectable concentration of virus at which &gt;95% of all replicates test positive (LoD) was 288 copies/mL. The results for the contrived clinical evaluation are shown in Table 1. 19/20 (≥95%) low positive specimens (1-2xLoD) and 20/20 (100%) moderate positive specimens (3-5x LoD) were detected demonstrating the functionality of the test.

Conclusion
VIASURE Monkeypox virus Real Time PCR Reagents for BD MAX™ System provides results in a short time and requires less than one minute of hands-on time per sample due to integration of fully automated extraction and thermocycling steps.
An additional clinical testing of positive natural clinical specimens must be performed to complete evidence that this product is for the purpose and the population for which it is intended.
B-270 Validation of a New Molecular Assay for the Detection and Quantification of Hepatitis B Load in Plasma
Authors: Cortés-Portero I , Dehesa-García B , García-Murillo A , García-Manrique B 
Published: September 2023

Abstract

Background
Hepatitis B virus (HBV) is an enveloped DNA virus and is the most common worldwide cause of viral hepatitis in humans. It is primarily transmitted through blood or blood products. Although the diagnosis of hepatitis viral infections is usually performed with serological markers in blood, this technique has limitations and therefore the diagnosis of the disease by qPCR allows an improvement in the diagnosis of the infection, therapeutic decision making and evaluation of the response to treatment. Therefore, CerTest Biotec has developed a quantitative real-time PCR assay for the detection and quantification of hepatitis B virus in plasma samples.

Methods
Plasma-EDTA samples characterized as positive or negative and quantified in IU/ml were analyzed. The selected samples were collected from infants and adults of both sexes between September 2019 and August 2020. The origin of the population is diverse, with the majority being European. For comparative analysis, the cobas® HBV molecular assay (Roche Diagnostics) using the cobas® 8800 system was used. VIASURE analysis was performed using the following workflow: the MagDEA® Dx SV automated extraction method with magLEAD® 12gC and CFX96™ Opus Real-Time PCR Detection System (Bio-Rad).

Results
A total of 210 clinical EDTA plasma samples were tested, of which 110 were positive for hepatitis B virus and 100 were negative. The comparative analysis determined a sensitivity and specificity &gt;99%. In addition, the positive predictive value (PPV) and negative predictive value were &gt;99%. As for quantification, an R2 correlation value of 0.9095 was obtained.

Conclusion
This clinical evaluation demonstrated that the VIASURE HBV q Real Time PCR Detection Kit is a good tool for the clinical diagnosis of the virus as it has good sensitivity and specificity. No false positive or false negative results were observed. Moreover, the correlation of the quantification with the reference method gives a value of R2 &gt; 0.9. Therefore, clinical diagnosis of hepatitis B virus by real-time PCR can encompass the limitations of the current diagnostic technique by serology.
B-273 Lymphogranuloma Venereum: Validation of a Real-Time PCR Kit for Its Rapid Diagnosis
Authors: Peris M , Milagro A , Martínez N F , Algara O , Martínez-Mateos D , Dehesa-García B , García-Manrique B , Rezusta A 
Published: September 2023

Abstract

Background
Chlamydia trachomatis serovars L1, L2 and L3 are responsible for lymphogranuloma venereum (LGV), a sexually transmitted infection that causes ulcerated lesions and adenopathy. Following the procedures in Clinical Microbiology recommendations, rectal samples positive for C. trachomatis are processed to detect the LGV-producing serovars in order to provide appropriate treatment. Since European regulations require for CE and IVD certification that an in vitro diagnostic product must be validated by an external body, the aim of this study was to validate the VIASURE C. trachomatis (LGV) Real Time PCR Detection Kit from Certest Biotec SL, using DNAs characterized as positive or negative for LGV extracted from clinical samples previously analyzed in the Laboratory.

Methods
A total of 214 DNAs from clinical samples collected between January 2021 and November 2022 were analyzed. Based on the initial diagnosis performed with the Allplex™ Genital ulcer Assay (Seegene®), 37 samples were positive for LGV and 177 were negative. The use of all data and samples was approved by the Aragon research ethics committee (CEICA) (PI20/426). The clinical sensitivity and specificity of the kit and the positive and negative predictive values (95% CI) were calculated with MetaDisc 1.4 software.

Results
Results are summarized in Table 1.

Conclusion
The comparative analysis of the “VIASURE C. trachomatis (LGV) Real Time PCR Detection Kit” proved to be as sensitive and specific as the reference molecular assay. The main advantage is that VIASURE products feature a stabilized, ready-to-use format, reducing the number of time-consuming laboratory steps, avoiding possible contamination and allowing transport and storage at room temperature.