Next-Generation Sequencing in High-Sensitive Detection of Mutations in Tumors
Next-generation sequencing (NGS) technologies have come of age as preferred technologies for screening of genomic variants of pathologic and therapeutic potential. Because of their capability for high-throughput and massively parallel sequencing, they can screen for a variety of genomic changes in multiple samples simultaneously. This has made them platforms of choice for clinical testing of solid tumors and hematological malignancies. Consequently, they are increasingly replacing conventional technologies, such as Sanger sequencing and pyrosequencing, expression arrays, real-time PCR, and fluorescence in situ hybridizatio...
Source: Journal of Molecular Diagnostics - May 28, 2020 Category: Pathology Authors: Rajesh R. Singh Tags: Review Source Type: research

Next-Generation Sequencing in High-Sensitive Detection of Mutations in Tumors: Challenges, Advances and Applications
Next-generation sequencing (NGS) technologies have come of age as preferred technologies for screening of genomic variants of pathologic and therapeutic potential. Owing to their capability for high throughput and massively parallel sequencing, they can screen for a variety of genomic changes in multiple samples simultaneously. This has made them platforms of choice for clinical testing of solid tumors and hematological malignancies. Consequently, they are increasingly replacing conventional technologies such as Sanger sequencing and Pyrosequencing, expression arrays, real-time polymerase chain reaction and fluorescence in...
Source: Journal of Molecular Diagnostics - May 28, 2020 Category: Pathology Authors: Rajesh R. Singh Source Type: research

Long molecule sequencing: a new approach for identification of clinically significant DNA variants in alpha and beta thalassemia carriers
Currently, carrier testing for thalassemia requires the application of different molecular tests to provide an accurate genotype. As an alternative methodology, we evaluated long molecule sequencing (LMS) on the PacBio Sequel platform for genotyping carriers of alpha ( α) or beta (β) thalassemia. Multiplex long PCR was used to generate representative amplicons for the α (HBA1/2) and β (HBB) gene loci. Following LMS, circular consensus sequencing reads were aligned to the hg19 reference genome and variants called using Free-Bayes software. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 28, 2020 Category: Pathology Authors: Liangpu Xu, Aiping Mao, Hui Liu, Baoheng Gui, Kwong Wai Choy, Hailong Huang, Qian Yu, Xiaojie Zhang, Meihuan Chen, Na Lin, Lingji Chen, Jin Han, Yan Wang, Min Zhang, Xiaoyu Li, Deqin He, Yuan Lin, Jianguang Zhang, David S. Cram, Hua Cao Source Type: research

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage
Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR) –associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. (Source...
Source: Journal of Molecular Diagnostics - May 26, 2020 Category: Pathology Authors: Haipo Xu, Xiaolong Zhang, Zhixiong Cai, Xiuqing Dong, Geng Chen, Zhenli Li, Liman Qiu, Lei He, Bin Liang, Xiaolong Liu, Jingfeng Liu Tags: Regular article Source Type: research

An isothermal method for sensitive detection of Mycobacterium tuberculosis complex using CRISPR/Cas12a cis- and trans-cleavage
Tuberculosis is one of the most serious infectious diseases resulting in lethal death worldwide. Traditional detection methods are still not enough to meet the clinical requirements of rapid diagnosis, high specificity and sensitivity. Fast, sensitive and accurate detection of mycobacterium tuberculosis (MTB) is an urgent need for the treatment and control of tuberculosis disease. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas12a) exhibits strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans-cleavage) after specific recognition of target sequenc...
Source: Journal of Molecular Diagnostics - May 26, 2020 Category: Pathology Authors: Haipo Xu, Xiaolong Zhang, Zhixiong Cai, Xiuqing Dong, Geng Chen, Zhenli Li, Liman Qiu, Lei He, Bin Liang, Xiaolong Liu, Jingfeng Liu Tags: Regular Article Source Type: research

Diagnostic Accuracy of Presurgical Staphylococcus aureus PCR Assay Compared with Culture and Post-PCR Implementation Surgical Site Infection Rates
Nasal colonization with Staphylococcus aureus is a well-referenced risk factor for postoperative surgical site infections (SSIs). Our health care system that performs>40,000 surgeries per year assessed both the diagnostic accuracy of the BD MAX StaphSR assay (MAX StaphSR), a PCR-based test that detects and differentiates S. aureus and methicillin-resistant S. aureus (MRSA), compared with our standard of care culture and the subsequent clinical impact on SSIs 1 year after implementation. In addition, residual specimens were tested by broth-enriched culture. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 22, 2020 Category: Pathology Authors: Giannoula S. Tansarli, Lindsay LeBlanc, Dianne B. Auld, Kimberle C. Chapin Tags: Regular article Source Type: research

Diagnostic accuracy of pre-surgical Staphylococcus aureus PCR assay compared to culture and post-PCR implementation surgical site infection rates
Nasal colonization with S. aureus is a well referenced risk factor for post-operative surgical site infections (SSIs). Our healthcare system that performs>40,000 surgeries per year assessed both the diagnostic accuracy of the BD MAX StaphSR (MAX StaphSR) assay, a PCR-based test that detects and differentiates Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), compared to our standard of care culture and the subsequent clinical impact on SSIs one year after implementation. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 22, 2020 Category: Pathology Authors: Giannoula S. Tansarli, Lindsay LeBlanc, Dianne B. Auld, Kimberle C. Chapin Tags: Regular Article Source Type: research

Enzymatic and Chemical-Based Methods to Inactivate Endogenous Blood Ribonucleases for Nucleic Acid Diagnostics
There are ongoing research efforts into simple and low-cost point-of-care nucleic acid amplification tests (NATs) addressing widespread diagnostic needs in resource-limited clinical settings. Nucleic acid testing for RNA targets in blood specimens typically requires sample preparation that inactivates robust blood ribonucleases (RNases) that can rapidly degrade exogenous RNA. Most NATs rely on decades-old methods that lyse pathogens and inactivate RNases with high concentrations of guanidinium salts. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 21, 2020 Category: Pathology Authors: Andrew T. Bender, Benjamin P. Sullivan, Lorraine Lillis, Jonathan D. Posner Tags: Regular article Source Type: research

Enzymatic and chemical-based methods to inactivate endogenous blood ribonucleases for nucleic acid diagnostics
There are ongoing research efforts into simple and low-cost point-of-care (POC) nucleic acid amplification tests (NATs) addressing widespread diagnostic needs in resource-limited clinical settings. Nucleic acid testing for RNA targets in blood specimens typically requires sample preparation that inactivates robust blood ribonucleases (RNases) that can rapidly degrade exogenous RNA. Most NATs rely on decades-old methods that lyse pathogens and inactivate RNases with high concentrations of guanidinium salts. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 21, 2020 Category: Pathology Authors: Andrew T. Bender, Benjamin P. Sullivan, Lorraine Lillis, Jonathan D. Posner Source Type: research

Laboratories and Pandemic Preparedness
The coronavirus pandemic of 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 was first detected in Wuhan, China, in December 2019 and spread globally during the early part of 2020. As of May 11, 2020,>4 million cases have been confirmed worldwide, with>1.3 million cases in the United States (https://coronavirus.jhu.edu/map.html, last accessed May 11, 2020). The United States now has the most confirmed cases and confirmed deaths worldwide. The key to slowing the spread of this disease is widespread testing so that patients can be quickly identified and isolated. (...
Source: Journal of Molecular Diagnostics - May 16, 2020 Category: Pathology Authors: Karen L. Kaul Tags: Guest editorial Source Type: research

Laboratories and pandemic preparedness: A framework for collaboration and oversight
The coronavirus pandemic of 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 was first detected in Wuhan, China in December 2019 and spread globally during the early part of 2020. As of May 11, 2020, over 4 million cases have been confirmed worldwide, with over 1.3 million cases in the United States (https://coronavirus.jhu.edu/map.html, last accessed May 11, 2020). The United States now has the most confirmed cases and confirmed deaths worldwide. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 16, 2020 Category: Pathology Authors: Karen L. Kaul Tags: Guest Editorial Source Type: research

Detection of Microsatellite Instability from Circulating Tumor DNA by Targeted Deep  Sequencing
Currently, microsatellite instability (MSI) detection is limited to tissue samples with sufficient tumor content. Detection of MSI from blood has been explored but confounded by low sensitivity due to limited circulating tumor DNA (ctDNA). We developed a next-generation sequencing –based algorithm, blood MSI signature enrichment analysis, to detect MSI from blood. Blood MSI signature enrichment analysis development involved three major steps. First, marker sites that can effectively distinguish high MSI (MSI-H) from microsatellite stable tumors were extracted. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 15, 2020 Category: Pathology Authors: Zhenghao Cai, Zhenxin Wang, Chenglin Liu, Dongtao Shi, Dapeng Li, Minhua Zheng, Han Han-Zhang, Analyn Lizaso, Jianxing Xiang, Juan Lv, Wenjun Wu, Zhihong Zhang, Zhou Zhang, Fei Yuan, Songbing He, Jing Sun Tags: Technical advance Source Type: research

Detection of microsatellite instability from circulating tumor DNA by targeted deep sequencing
In this study, we developed an NGS-based algorithm, bMSISEA (blood MSI signature enrichment analysis), to detect MSI from blood samples. BMSISEA development involved 3 major steps. First, marker sites that can effectively distinguish MSI-high (MSI-H) from microsatellite stable (MSS) tumors were extracted. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 15, 2020 Category: Pathology Authors: Zhenghao Cai, Zhenxin Wang, Chenglin Liu, Dongtao Shi, Dapeng Li, Minhua Zheng, Han Han-Zhang, Analyn Lizaso, Jianxing Xiang, Juan Lv, Wenjun Wu, Zhihong Zhang, Zhou Zhang, Fei Yuan, Songbing He, Jing Sun Tags: Technical Advance Source Type: research

Implementation of an Emergency Use Authorization Test During an Impending National Crisis
The laboratory response to the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic may be termed heroic. From the identification of the novel coronavirus to implementation of routine laboratory testing around the world to the development of potential vaccines, laboratories have played a critical role in the efforts to curtail this pandemic. In this brief report, we review our own effort at a mid-sized, rural, academic medical center to implement a molecular test for the virus; and, we share insights and lessons learned from that process which might be helpful in similar situations in the future. (...
Source: Journal of Molecular Diagnostics - May 14, 2020 Category: Pathology Authors: Joel A. Lefferts, Edward J. Gutmann, Isabella W. Martin, Wendy A. Wells, Gregory J. Tsongalis Tags: Perspectives Source Type: research

Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Is Comparable in Clinical Samples Preserved in Saline or Viral Transport Medium
As the coronavirus disease 2019 (COVID-19) pandemic sweeps across the world, the availability of viral transport medium (VTM) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. Given that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA has demonstrated stability, we posited that phosphate-buffered saline (PBS) may be a viable transport medium, as an alternative to VTM, for clinical real-time quantitative PCR (qPCR) testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 13, 2020 Category: Pathology Authors: Jared Radbel, Sugeet Jagpal, Jason Roy, Andrew Brooks, Jay Tischfield, Michael Sheldon, Christian Bixby, Dana Witt, Maria L. Gennaro, Daniel B. Horton, Emily S. Barrett, Jeffrey L. Carson, Reynold A. Panettieri, Martin J. Blaser Tags: Regular article Source Type: research

Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Is Comparable in Clinical Samples Preserved in Saline or Viral Transport Medium
As the coronavirus disease 2019 (COVID-19) pandemic sweeps across the world, the availability of viral transport medium (VTM) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. Given that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA has demonstrated stability, we posited that phosphate-buffered saline (PBS) may be a viable transport medium, as an alternative to VTM, for clinical real-time quantitative PCR (qPCR) testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 13, 2020 Category: Pathology Authors: Jared Radbel, Sugeet Jagpal, Jason Roy, Andrew Brooks, Jay Tischfield, Michael Sheldon, Christian Bixby, Dana Witt, Maria L. Gennaro, Daniel B. Horton, Emily S. Barrett, Jeffrey L. Carson, Reynold A. Panettieri, Martin J. Blaser Tags: Regular article Source Type: research

Detection of SARS-CoV-2 is comparable in clinical samples preserved in saline or viral transport media
As the COVID-19 pandemic sweeps across the world, the availability of viral transport media (VTM) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. Given that SARS-CoV-2 viral RNA has demonstrated stability, we posited that phosphate buffered saline (PBS) may be a viable transport medium, as an alternative to VTM), for clinical qPCR testing. We assessed the intra- and inter-individual reliability of SARS-CoV-2 qPCR in clinical endotracheal secretion samples transported in VTM or PBS, evaluating the stability of the RT-qPCR signal for three viral targets (N gene, ORF1ab, a...
Source: Journal of Molecular Diagnostics - May 13, 2020 Category: Pathology Authors: Jared Radbel, Sugeet Jagpal, Jason Roy, Andrew Brooks, Jay Tischfield, Michael Sheldon, Christian Bixby, Dana Witt, Maria Laura Gennaro, Daniel B. Horton, Emily S. Barrett, Jeffrey L. Carson, Reynold A. Panettieri, Martin J. Blaser Tags: Regular Article Source Type: research

Sputum Cell-Free DNA
This study established an improved sputum cell-free DNA (cfDNA) extraction method study and applied a super –amplification refractory mutation system to detect the EGFR mutation status in sputum cfDNA. The sputum sediments were used for cytology evaluation. The study included 102 lung adenocarcinoma patients; 65 patients (63.7%) were positive for EGFR mutations in tumor samples. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2020 Category: Pathology Authors: Zheng Wang, Lin Zhang, Lin Li, Xiaoguang Li, Yan Xu, Mengzhao Wang, Li Liang, Peng Jiao, Yuanming Li, Shurong He, Jun Du, Lei He, Min Tang, Mingjun Sun, Li Yang, Jing Di, Guanshan Zhu, Hong Shi, Dongge Liu Tags: Regular article Source Type: research

Application of Multiplex Bisulfite PCR –Ligase Detection Reaction–Real-Time Quantitative PCR Assay in Interrogating Bioinformatically Identified, Blood-Based Methylation Markers for Colorectal Cancer
This report demonstrates the ability of a multiplex bisulfite PCR –ligase detection reaction–real-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patient-derived cell-free DNAs. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2020 Category: Pathology Authors: Manny D. Bacolod, Aashiq H. Mirza, Jianmin Huang, Sarah F. Giardina, Philip B. Feinberg, Steven A. Soper, Francis Barany Tags: Regular article Source Type: research

Sputum Cell-free DNA: Valued Surrogate Sample for Detection of EGFR Mutation in Patients with Advanced Lung Adenocarcinoma
Sputum is common cytological sample type, but its potential use in EGFR mutation detection in patients with lung cancer is not fully evaluated. An improved sputum cfDNA extraction method was established in this study, and applied super-amplification refractory mutation system (SuperARMS) to detect the EGFR mutation status in sputum cfDNA. The sputum sediments were used for cytology evaluation. The study included 102 lung adenocarcinoma patients, in which 65 (63.7%) patients were positive for EGFR mutations in tumor samples. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2020 Category: Pathology Authors: Zheng Wang, Lin Zhang, Lin Li, Xiaoguang Li, Yan Xu, Mengzhao Wang, Li Liang, Peng Jiao, Yuanming Li, Shurong He, Jun Du, Lei He, Min Tang, Mingjun Sun, Li Yang, Jing Di, Guanshan Zhu, Hong Shi, Dongge Liu Source Type: research

Application of multiplex bisulfite PCR-LDR-qPCR assay in interrogating bioinformatically-identified, blood-based methylation markers for colorectal cancer
This report demonstrates the ability of a multiplex bisulfite PCR-LDR-qPCR assay to detect 7 methylated CpG markers (CRC- or colon-specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with roughly 3000 copies of fragmented peripheral blood DNA), and CRC patient-derived cell-free DNAs (cfDNAs). (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 11, 2020 Category: Pathology Authors: Manny D. Bacolod, Aashiq H. Mirza, Jianmin Huang, Sarah F. Giardina, Philip B. Feinberg, Steven A. Soper, Francis Barany Source Type: research

Expression of GNAS, TP53, and PTEN Improves the Patient Prognostication in Sonic Hedgehog (SHH) Medulloblastoma Subgroup
In this study, formalin-fixed, paraffin-embedded samples from 164 Brazilian medulloblastomas were evaluated, applying the 22-gene panel, and subclassified into the low and high expression of nine key medulloblastoma-related genes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 4, 2020 Category: Pathology Authors: Luciane S. da Silva, Bruna M. Man çano, Flávia E. de Paula, Mariana B. dos Reis, Gisele C. de Almeida, Marcus Matsushita, Carlos A. Junior, Adriane F. Evangelista, Fabiano Saggioro, Luciano N. Serafini, João N. Stavale, Suzana M.F. Malheiros, Matheus L Tags: Regular article Source Type: research

Recommendations for Clinical Warfarin Genotyping Allele Selection
The goal of the Association for Molecular Pathology (AMP) Clinical Practice Committee's AMP Pharmacogenomics (PGx) Working Group is to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 4, 2020 Category: Pathology Authors: Victoria M. Pratt, Larisa H. Cavallari, Andria L. Del Tredici, Houda Hachad, Yuan Ji, Lisa V. Kalman, Reynold C. Ly, Ann M. Moyer, Stuart A. Scott, Michelle Whirl-Carrillo, Karen E. Weck Tags: Special article Source Type: research

Recommendations for Clinical Warfarin Sensitivity Genotyping Allele Selection
The goal of the Association for Molecular Pathology (AMP) Clinical Practice Committee's AMP Pharmacogenomics (PGx) Working Group is to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 4, 2020 Category: Pathology Authors: Victoria M. Pratt, Larisa H. Cavallari, Andria L. Del Tredici, Houda Hachad, Yuan Ji, Lisa V. Kalman, Reynold C. Ly, Ann M. Moyer, Stuart A. Scott, Michelle Whirl-Carrillo, Karen E. Weck Tags: Special article Source Type: research

Recommendations for Clinical Warfarin Sensitivity Genotyping Allele Selection: A Report of the Association for Molecular Pathology and College of American Pathologists
The goals of the Association for Molecular Pathology (AMP) Clinical Practice Committee ’s AMP Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic (PGx) alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provide recommendations for a minimum panel o f variant alleles (“Tier 1”) and an extended panel of variant alleles (“Tier 2”) that will aid clinical laboratories when designing assays for PGx testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 4, 2020 Category: Pathology Authors: Victoria M. Pratt, Larisa H. Cavallari, Andria L. Del Tredici, Houda Hachad, Yuan Ji, Lisa V. Kalman, Reynold C. Ly, Ann M. Moyer, Stuart A. Scott, Michelle Whirl-Carrillo, Karen E. Weck Tags: Special Article Source Type: research

Expression of GNAS, TP53, and PTEN Improves the Patient Prognostication in SHH Medulloblastoma Subgroup
In this study, FFPE samples from 164 Brazilian medulloblastomas were evaluated applying the 22-gene panel and subclassified into the low and high expression of nine key medulloblastoma-related genes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 4, 2020 Category: Pathology Authors: Luciane Sussuchi da Silva, Bruna Minniti Man çano, Flávia Escremim de Paula, Mariana Bisarro dos Reis, Gisele Caravina de Almeida, Marcus Matsushita, Carlos Almeida Junior, Adriane Feijó Evangelista, Fabiano Saggioro, Luciano Neder Serafini, João Norb Tags: Regular Article Source Type: research

Development and Clinical Validation of Discriminatory Multitarget Digital Droplet PCR Assays for the Detection of Hot Spot KRAS and NRAS Mutations in Cell-Free DNA
Detection and quantification of tumor-derived KRAS and NRAS mutations in plasma cell-free DNA (cfDNA) holds great potential for cancer diagnostics and treatment response monitoring. Because of high sensitivity, specificity, robustness, and affordability, digital droplet PCR (ddPCR) is ideally suited for this application but requires discriminatory multiplexing when used as screening assay. We therefore designed, optimized, and clinically validated mutation-specific locked nucleic acid –based ddPCR assays for 14 commonly occurring KRAS and NRAS mutations and assembled these assays into seven discriminatory multitarget...
Source: Journal of Molecular Diagnostics - May 3, 2020 Category: Pathology Authors: Saskia Hussung, Marie Follo, Rhena F.U. Klar, Sandra Michalczyk, Kornelia Fritsch, Friederike Nollmann, Julian Hipp, Justus Duyster, Florian Scherer, Nikolas von Bubnoff, Melanie Boerries, Uwe Wittel, Ralph M. Fritsch Tags: Regular article Source Type: research

Development and clinical validation of discriminatory multi-target digital droplet PCR assays for the detection of hot spot KRAS and NRAS mutations in cell-free DNA
Detection and quantification of tumor-derived KRAS and NRAS mutations in cell-free plasma DNA (cfDNA) holds great potential for cancer diagnostics and treatment response monitoring. Because of high sensitivity, specificity, robustness and affordability, digital droplet PCR (ddPCR) is ideally suited for this application but requires discriminatory multiplexing when used as screening assay. We therefore designed, optimized and clinically validated mutation-specific locked nucleic acid (LNA)-based ddPCR assays for 14 commonly occurring KRAS and NRAS mutations and assembled these assays into 7 discriminatory multi-target scree...
Source: Journal of Molecular Diagnostics - May 3, 2020 Category: Pathology Authors: Saskia Hussung, Marie Follo, Rhena F.U. Klar, Sandra Michalczyk, Kornelia Fritsch, Friederike Nollmann, Julian Hipp, Justus Duyster, Florian Scherer, Nikolas von Bubnoff, Melanie Boerries, Uwe Wittel, Ralph M. Fritsch Tags: Regular Article Source Type: research

Abstracts of the 2nd AMP Europe Congress
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2020 Category: Pathology Source Type: research

Author Index
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2020 Category: Pathology Source Type: research

Loop-Primer Endonuclease Cleavage –Loop-Mediated Isothermal Amplification Technology for Multiplex Pathogen Detection and Single-Nucleotide Polymorphism Identification
This study introduces loop-primer endonuclease cleavage (LEC) –LAMP, a singleplex or multiplex LAMP technology with single-base specificity for variable SNP identification. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2020 Category: Pathology Authors: Owen Higgins, Terry J. Smith Tags: Regular article Source Type: research

Detection and Quantification of Chimeric Antigen Receptor Transgene Copy Number by Droplet Digital PCR versus Real-Time PCR
In this study, key performance parameters of the ddPCR and real-time PCR methods were assessed, including linearity, detection range, the lower limit of detection, repeatability, reproducibility, and accuracy, using a series of gradient diluted standards and clinical peripheral blood samples from CAR T-cell patients. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2020 Category: Pathology Authors: Yaoyao Lou, Caixia Chen, Xiaolu Long, Jia Gu, Min Xiao, Di Wang, Xiaoxi Zhou, Tongjuan Li, Zhenya Hong, Chunrui Li, Jianfeng Zhou, Liting Chen Tags: Regular article Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2020 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2020 Category: Pathology Source Type: research

Treatment response monitoring in patients with advanced malignancies using cell-free SHOX2 and SEPT9 DNA methylation in blood: An observational prospective study
Patients with incurable cancer usually receive palliative treatment with significant toxicity and limited efficacy. Methylation analysis of circulating cell-free DNA in blood from cancer patients represents a promising approach for minimally invasive, real-time monitoring of treatment response. SHOX2 and SEPT9 methylation was analyzed in N=8,865 malignant and N=746 normal adjacent tissues across 33 different malignancies from The Cancer Genome Atlas. Furthermore, we performed quantitative SHOX2 and SEPT9 circulating cell-free DNA methylation analysis in plasma obtained before and consecutively during treatment from prospec...
Source: Journal of Molecular Diagnostics - April 30, 2020 Category: Pathology Authors: Luka de Vos, Maria Jung, Ruth-Miriam Koerber, Emma Grace Bawden, Tobias A.W. Holderried, J örn Dietrich, Friedrich Bootz, Peter Brossart, Glen Kristiansen, Dimo Dietrich Source Type: research

Highly Sensitive and Specific Detection and Serotyping of Five Prevalent Salmonella Serovars by Multiple Cross-Displacement Amplification
Salmonella is a common cause of foodborne disease worldwide, including Australia. More than 85% of outbreaks of human salmonellosis in Australia were caused by five Salmonella serovars. Rapid, accurate, and sensitive identification of Salmonella serovars is vital for diagnosis and public health surveillance. Recently, an isothermal amplification technique, termed multiple cross-displacement amplification (MCDA), has been employed to detect Salmonella at the species level. Herein, seven MCDA assays were developed and evaluated for rapid detection and differentiation of the five most common Salmonella serovars in Australia: ...
Source: Journal of Molecular Diagnostics - April 28, 2020 Category: Pathology Authors: Xiaomei Zhang, Michael Payne, Qinning Wang, Vitali Sintchenko, Ruiting Lan Tags: Regular article Source Type: research

Low-Pass Genome Sequencing
DNA copy number variants (CNVs) account for approximately 300 Mb of sequence variation in the normal human genome. Significant numbers of pathogenic CNVs contribute toward human genetic disorders. Recent studies suggest a higher diagnostic and clinical significance of low-pass genome sequencing (LP-GS) compared with chromosomal microarrays (CMAs). The performance metrics of the 5X LP-GS was compared with CMA to validate a low-cost and high-throughput method. LP-GS test performed on 409 samples (including 78 validation and 331 clinical) was evaluated using American College of Medical Genetics and Genomics guidelines. (Sourc...
Source: Journal of Molecular Diagnostics - April 25, 2020 Category: Pathology Authors: Alka Chaubey, Suresh Shenoy, Abhinav Mathur, Zeqiang Ma, C. Alexander Valencia, Babi R. Reddy Nallamilli, Edward Szekeres, Leah Stansberry, Ruby Liu, Madhuri R. Hegde Tags: Regular article Source Type: research

Low Pass-Genome Sequencing: Validation and diagnostic utility from 409 clinical cases of low-pass genome sequencing for the detection of copy number variants (CNVs) to replace constitutional microarray
DNA copy-number variants (CNVs) account for approximately 300 Mb of sequence variation in the normal human genome. Significant numbers of pathogenic CNVs contribute towards human genetic disorders. Recent studies suggest a higher diagnostic and clinical significance of low-pass genome sequencing (LP-GS) compared to microarrays (CMA). The performance metrics of the 5X LP-GS was compared to CMA to validate a low-cost and high throughput method. LP-GS test performed on 409 samples (including 78 validation and 331 clinical) were evaluated using American College of Medical Genetics and Genomics guidelines. (Source: Journal of M...
Source: Journal of Molecular Diagnostics - April 25, 2020 Category: Pathology Authors: Alka Chaubey, Suresh Shenoy, Abhinav Mathur, Zeqiang Ma, C. Alexander Valencia, Babi Ramesh Reddy Nallamilli, Edward Szekeres, Leah Stansberry, Ruby Liu, Madhuri R. Hegde Tags: Regular Article Source Type: research

Mass Spectrometry –Based Method Targeting Ig Variable Regions for Assessment of Minimal Residual Disease in Multiple Myeloma
We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 14, 2020 Category: Pathology Authors: Carlo O. Martins, Sarah Huet, San S. Yi, Maria S. Ritorto, Ola Landgren, Ahmet Dogan, Jessica R. Chapman Source Type: research

Long-Read Nanopore Sequencing Validated for Human Leukocyte Antigen Class I Typing in Routine Diagnostics
Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. (Sour...
Source: Journal of Molecular Diagnostics - April 14, 2020 Category: Pathology Authors: Benedict M. Matern, Timo I. Olieslagers, Mathijs Groeneweg, Burcu Duygu, Lotte Wieten, Marcel G.J. Tilanus, Christina E.M. Voorter Tags: Regular article Source Type: research

Continual Improvement of the Reliability of EML4-ALK Rearrangement Detection in Non –Small-Cell Lung Cancer
The results of EML4-ALK testing are critical to manage ALK inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 14, 2020 Category: Pathology Authors: Rongxue Peng, Rui Zhang, Jiawei Zhang, Ping Tan, Yanxi Han, Kuo Zhang, Guigao Lin, Jiehong Xie, Jinming Li Source Type: research

Long-read nanopore sequencing validated for HLA-class I typing in routine diagnostics
Matching of HLA gene polymorphisms by high-resolution DNA sequence analysis is gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications such as Human Leukocyte Antigen (HLA) typing for HLA matching of donor and recipient in the transplantation setting. (Source: J...
Source: Journal of Molecular Diagnostics - April 14, 2020 Category: Pathology Authors: Benedict M. Matern, Timo I. Olieslagers, Mathijs Groeneweg, Burcu Duygu, Lotte Wieten, Marcel G.J. Tilanus, Christina E.M. Voorter Source Type: research

Mass spectrometry-based method targeting Ig variable regions for assessment of minimal residual disease in multiple myeloma
We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 14, 2020 Category: Pathology Authors: Carlo O. Martins, Sarah Huet, San S. Yi, Maria Stella Ritorto, Ola Landgren, Ahmet Dogan, Jessica R. Chapman Source Type: research

Continual improvement of the reliability of EML4-ALK rearrangement detection in non-small cell lung cancer: a long-term comparison of ALK detection in China
The results of EML4-ALK testing are critical to manage ALK inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China since 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating 3 different variants and wild-type samples were collected between 2015 and 2019. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 14, 2020 Category: Pathology Authors: Rongxue Peng, Rui Zhang, Jiawei Zhang, Ping Tan, Yanxi Han, Kuo Zhang, Guigao Lin, Jiehong Xie, Jinming Li Source Type: research

Clinical Utility of a High-Resolution Melting Test for Screening Numerical Chromosomal Abnormalities in Recurrent Pregnancy Loss
We examined 765 products-of-conception samples with known karyotypes retrospectively using the HRM test, which showed high technical sensitivity (96.1%) and specificity ( 96.3%) as well as a high positive predictive value (95.9%) for the screening of chromosomal abnormalities. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 9, 2020 Category: Pathology Authors: Yulin Zhou, Wenyan Xu, Yancheng Jiang, Zhongmin Xia, Haixia Zhang, Xiaolu Chen, Zengge Wang, Yunsheng Ge, Qiwei Guo Tags: Regular article Source Type: research

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2
The coronavirus disease 2019 (COVID-19) pandemic now has>2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 7, 2020 Category: Pathology Authors: Gun-Soo Park, Keunbon Ku, Seung-Hwa Baek, Seong-Jun Kim, Seung Il Kim, Bum-Tae Kim, Jin-Soo Maeng Tags: Regular article Source Type: research

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2
Epidemics of coronavirus disease 2019 (COVID-19) now have>100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. Herein, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19, were developed and evaluated. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 7, 2020 Category: Pathology Authors: Gun-Soo Park, Keunbon Ku, Seung-Hwa Baek, Seong-Jun Kim, Seung Il Kim, Bum-Tae Kim, Jin-Soo Maeng Tags: Regular article Source Type: research

Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2
Epidemics of Coronavirus Disease 2019 (COVID-19) now have more than 100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by RT-qPCR methods, but the capacity of RT-qPCR methods is limited by its requirement of high-level facilities and instruments. Here, we developed and evaluated RT-LAMP assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human Coronaviruses was not observed. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 7, 2020 Category: Pathology Authors: Gun-Soo Park, Keunbon Ku, Seung-Hwa Baek, Seong-Jun Kim, Seung Il Kim, Bum-Tae Kim, Jin-Soo Maeng Source Type: research