CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Molecular Pathology, Diagnostics and Therapeutics: A Story of Success in 2022.

Molecular pathology, diagnostics and therapeutics are three closely related topics of critical importance in medical research and clinical practice [...].

Forty Years of Molecular Diagnostics for Infectious Diseases.

Nearly 40 years have elapsed since the invention of the PCR, with its extremely sensitive and specific ability to detect nucleic acids via in vitro enzyme-mediated amplification. In turn, more than 2 years have passed since the onset of the coronavirus disease 2019 (COVID-19) pandemic, during which time molecular diagnostics for infectious diseases have assumed a larger global role than ever before. In this context, we review broadly the progression of molecular techniques in clinical microbiology, to their current prominence. Notably, these methods now entail both the detection and quantification of microbial nucleic acids, along with their sequence-based characterization. Overall, we seek to provide a combined perspective on the techniques themselves, as well as how they have come to shape health care at the intersection of technologic innovation, pathophysiologic knowledge, clinical/laboratory logistics, and even financial/regulatory factors.

Trends in molecular diagnostics: 12th European Meeting on Molecular Diagnostics, Noordwijk aan Zee, the Netherlands, 12-14 October 2022.

This report provides an overview of the highlights of the 12th European Meeting on Molecular Diagnostics held in Noordwijk aan Zee, The Netherlands, 12-14 October 2022. This 3-day conference covered many relevant topics in the field of molecular diagnostics in humans i.e. oncology, infectious diseases, laboratory medicine, pharmacogenetics, pathology, and preventive medicine. Other relevant topics included quality management, laboratory automation, diagnostic preparedness, and lessons learned from the COVID pandemic. More than 400 participants, the majority coming from European countries, attended the meeting. Besides high-quality scientific presentations, more than 40 diagnostic companies presented their latest innovations, altogether in an informal and inspiring ambiance.

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

SARS-CoV-2 Molecular Diagnostics in China.

The COVID-19 pandemic remains a significant problem involving health systems worldwide. Several diagnostic methods are reported for detecting the coronavirus in clinical, research, and public health laboratories. rRT-PCR is considered the gold standard; however, as it required skilled personnel and special equipment, rapid antigen tests have been developed and used as first-line screening. The serologic testing of antibodies can also be used to enhance the detection sensitivity and accuracy, which are used to assess the overall infection rate. This review summarizes the molecular techniques and serologic assays widely used in China and discusses the advantages and disadvantages of these techniques.

An Overview of SARS-CoV-2 Molecular Diagnostics in Europe.

The COVID-19 pandemic has led to the rapid development of a plethora of molecular diagnostic assays with real-time polymerase chain reaction (RT-PCR) at the forefront. In this review, we will discuss the history and utility of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) molecular diagnostics and the associated current and future regulatory process in Europe. We will assess the performance characteristics of a range of the most common SARS-CoV-2 molecular tests currently used in Europe with a focus on as rapid molecular platforms, stand-alone RT-PCR kits, the role of low-throughput and high-throughput end-to-end testing platforms, and the rapidly evolving field of SARS-CoV-2 variant of concern identification.

Considerations for the selection of tests for SARS-CoV-2 molecular diagnostics.

During the course of 2020, the outbreak of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spread rapidly across the world. Clinical diagnostic testing for SARS-Cov-2 infection has relied on the real-time Reverse Transcriptase Polymerase Chain Reaction and is considered the gold standard assay. Commercial vendors and laboratories quickly mobilised to develop diagnostic tests to detect the novel coronavirus, which was fundamentally important in the pandemic response. These SARS-Cov-2 assays were developed in line with the Food Drug Administration-Emergency Use Authorization guidance. Although new tests are continuously being developed, information about SARS-CoV-2 diagnostic molecular test accuracy has been limited and at times controversial. Therefore, the analytical and clinical performance of SARS-CoV-2 test kits should be carefully considered by the appropriate regulatory authorities and evaluated by independent laboratory validation. This would provide improved end-user confidence in selecting the most reliable and accurate diagnostic test. Moreover, it is unclear whether some of these rapidly developed tests have been subjected to rigorous quality control and assurance required under good manufacturing practice. Variable target gene regions selected for currently available tests, potential mutation in target gene regions, non-standardized pre-analytic phase, a lack of manufacturer independent validation data all create difficulties in selecting tests appropriate for different countries and laboratories. Here we provide information on test criteria which are important in the assessment and selection of SARS-CoV-2 molecular diagnostic tests and outline the potential issues associated with a proportion of the tests on the market.

SARS-CoV-2 Delta Variant N Gene Mutations Reduce Sensitivity to the TaqPath COVID-19 Multiplex Molecular Diagnostic Assay.

As the SARS-CoV-2 virus evolves, mutations may result in diminished sensitivity to qRT-PCR diagnostic assays. We investigated four polymorphisms circulating in the SARS-CoV-2 Delta lineage that result in N gene target failure (NGTF) on the TaqPath COVID-19 Combo Kit. These mutations were detected from the SARS-CoV-2 genome sequences that matched with the diagnostic assay results of saliva specimens. Full length N genes from the samples displaying NGTF were cloned into plasmids and assayed using three SARS-CoV-2 qRT-PCR assays. These constructs resulted in reduced sensitivity to the TaqPath COVID-19 Combo Kit compared to the controls (mean Ct differences of 3.06, 7.70, 12.46, and 14.12), but were detected equivalently on the TaqPath COVID-19 Fast PCR Combo 2.0 or CDC 2019_nCoV_N2 assays. This work highlights the importance of genomic sequencing to monitor circulating mutations and provide guidance in improving diagnostic assays.

Managing Viral Emerging Infectious Diseases via current Molecular Diagnostics in the Emergency Department: the Tricky Cases.

INTRODUCTION: Emerging infectious diseases' diagnosis has been a major problem in most hospitals and other senior care facilities, especially for the current Coronavirus Disease 2019 (COVID-19). The various clinical manifestations, and the several radiology and laboratory data combined with the misleading test results for identifying the virus, are responsible for certain misdiagnoses, especially for suspected cases that visit the emergency department and require urgent management and further treatment. AREAS COVERED: The major challenges for emerging infectious diseases' molecular diagnosis are being described here on a great scale, and, finally, strategies for a precise and on-the-spot molecular diagnosis are thoroughly discussed. Related literature was searched using the PubMed, Science Direct, and EMBASE databases published until May 2022 on the general information for viral infections and relevant false test results. EXPERT OPINION: Emerging diseases' molecular diagnosis via current common diagnostic assays seems to be extremely tricky, and front-line physicians and other senior care facilities should be able to recognize some falsely diagnosed cases or even prevent their existence. Further biotechnologic revolution concerning viral molecular diagnostics will be evident in the near future, thus new methods' limitations should be highlighted to physicians from the very beginning of their performances and wide utilization.

Automated sample-to-answer centrifugal microfluidic system for rapid molecular diagnostics of SARS-CoV-2.

Testing for SARS-CoV-2 is one of the most important assets in COVID-19 management and mitigation. At the onset of the pandemic, SARS-CoV-2 testing was uniquely performed in central laboratories using RT-qPCR. RT-qPCR relies on trained personnel operating complex instrumentation, while time-to-result can be lengthy (e.g., 24 to 72 h). Now, two years into the pandemic, with the surge in cases driven by the highly transmissible Omicron variant, COVID-19 testing capabilities have been stretched to their limit worldwide. Rapid antigen tests are playing an increasingly important role in quelling outbreaks by expanding testing capacity outside the realm of clinical laboratories. These tests can be deployed in settings where repeat and rapid testing is essential, but they often come at the expense of limited accuracy and sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a number of advantages to SARS-CoV-2 testing in standard laboratories and at the point-of-need. In contrast to RT-qPCR, RT-LAMP is performed at a constant temperature, which circumvents the need for thermal cycling and translates into a shorter analysis time (e.g., <1 h). In addition, RT-LAMP is compatible with colorimetric detection, facilitating visualization and read-out. However, even with these benefits, RT-LAMP is not yet clinically deployed at its full capacity. Lack of automation and integration of sample preparation, such as RNA extraction, limits the sensitivity and specificity of the method. Furthermore, the need for cold storage of reagents complicates its use at the point of need. The developments presented in this work address these limitations: We describe a fully automated SARS-CoV-2 detection method using RT-LAMP, which also includes up-front lysis and extraction of viral RNA, performed on a centrifugal platform with active pneumatic pumping, a disposable, all-polymer-based microfluidic cartridge and lyophilized reagents. We demonstrate that the limit of detection of the RT-LAMP assay itself is 0.2 copies per µL using N and E genes as target sequences. When combined with integrated RNA extraction, the assay sensitivity is 0.5 copies per µL, which is highly competitive to RT-qPCR. We tested the automated assay using 12 clinical swab specimens from patients and were able to distinguish positive and negative samples for SARS-CoV-2 within 60 min, thereby obtaining 100% agreement with RT-qPCR results.

RT-LAMP-Based Molecular Diagnostic Set-Up for Rapid Hepatitis C Virus Testing.

Hepatitis C virus (HCV) infections occur in approximately 3% of the world population. The development of an enhanced and extensive-scale screening is required to accomplish the World Health Organization's (WHO) goal of eliminating HCV as a public health problem by 2030. However, standard testing methods are time-consuming, expensive, and challenging to deploy in remote and underdeveloped areas. Therefore, a cost-effective, rapid, and accurate point-of-care (POC) diagnostic test is needed to properly manage the disease and reduce the economic burden caused by high case numbers. Herein, we present a fully automated reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based molecular diagnostic set-up for rapid HCV detection. The set-up consists of an automated disposable microfluidic chip, a small surface heater, and a reusable magnetic actuation platform. The microfluidic chip contains multiple chambers in which the plasma sample is processed. The system utilizes SYBR green dye to detect the amplification product with the naked eye. The efficiency of the microfluidic chip was tested with human plasma samples spiked with HCV virions, and the limit of detection observed was 500 virions/mL within 45 min. The entire virus detection process was executed inside a uniquely designed, inexpensive, disposable, and self-driven microfluidic chip with high sensitivity and specificity.

Molecular diagnostics of SARS-CoV-2: Findings of an international survey.

BACKGROUND: In the current COVID-19 pandemic, early and rapid diagnosis of potentially infected and contagious individuals enables containment of the disease through quarantine and contact tracing. The rapid global expansion of these diagnostic testing services raises questions concerning the current state of the art with regard to standardization of testing and quality assessment practices. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices used for SARS-CoV-2 diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) initiated a survey among international laboratories performing molecular genetic detection of SARS-CoV-2. Questions on quality assurance, variant testing, sequencing and the transmission of findings were included in the survey. RESULTS: A total of 273 laboratories from 49 countries participated in the survey. The majority of the participating laboratories (92.2%) use reverse transcriptase polymerase chain reaction (RT-PCR). The majority of participating laboratories do not conduct testing to identify SARS CoV-2 variants. Participation in external quality assessment programs was reported by the majority of laboratories, however, 33.2% of the laboratories reported not participating in external quality assurance programmes. CONCLUSIONS: Based on the survey, molecular diagnostic methods for SARS-CoV-2 detection are clearly not standardized across different countries and laboratories. The survey found an array of responses in regard to sample preparation, collection, processing and reporting of results. This work suggests quality assurance is insufficiently performed by diagnostic laboratories conducting SARS-CoV-2 testing.

Challenges and experiences of the SARS-CoV-2 pandemic in molecular diagnostics / A SARS-CoV-2-járvány kihívásai és tapasztalatai a molekuláris diagnosztikában

Összefoglaló. Bár a SARS-CoV-2-pandémia próbára tette a diagnosztikus kapacitásokat, számos hasznos tapasztalattal is szolgált, melyek alacsonyabb mintaszám mellett nem lettek volna levonhatók. Míg korábban a PCR-vizsgálatok jellemzoen diagnosztikus, illetve kvantitatív követési célokat szolgáltak, a járvány során többségbe kerültek a szuro- és (kezdetben) a felszabadító vizsgálatok. Jól követheto volt, hogy a tesztek piacra juttatásának eroltetett üteme sokszor nem tette lehetové a teljesen kiforrott koncepciók létrehozását. Tekintettel arra, hogy a molekuláris diagnosztika során nem teljes vírusgenomokat, hanem célszakaszokat mutatunk ki, amelyek aránya a fertozés egyes szakaszaiban nem feltétlenül állandó, egyre valószínubb, hogy nem azonos célgének a legmegfelelobbek diagnosztikus, szuro- és felszabadító vizsgálatokhoz. A nagy mennyiségu, aspecifikusan végzett vizsgálat még kiváló fajlagosság mellett is a pozitív prediktív érték csökkenéséhez vezethet, amennyiben a fertozés tényleges prevalenciája a vizsgálati csoportban alacsony. Munkánkban megkíséreljük irodalmi és saját adatok felhasználásával összefoglalni az elmúlt két év fontosabb diagnosztikus tapasztalatait a teljesség igénye nélkül. Orv Hetil. 2021; 162(52): 2071-2078. Summary. Although the SARS-CoV-2 pandemic has been a great challenge for the diagnostic capacities, it also proved to be a unique source of experience. While previously PCR tests had overwhelmingly been used for targeted diagnostic and quantitative follow-up testing, screening and (initially) release tests became far more prevalent during the pandemic. It was well to be seen that the forced pace of bringing tests to market often gave way to not fully mature concepts. The PCR method is based on the detection of sequences, the proportions of which are likely to alter throughout the course of the disease. It is becoming increasingly clear that different target genes might be the best suitable for diagnostic, screening and release testing. Even with specific assays, an unprecedentedly high number of tests might result in the inflation of the positive predictive value, when the true prevalence of the infection remains very low among the tested individuals. Here we try to summarize some of the potentially most relevant diagnostic conclusions of the pandemic so far according to our own data and the literature. Orv Hetil. 2021; 162(52): 2071-2078.

Molecular Diagnostic System Using Engineered Fusion Protein-Conjugated Magnetic Nanoparticles.

To effectively control the spread of new infectious diseases, there is a need for highly sensitive diagnostic methods to detect viral nucleic acids rapidly. This study outlines a universal and simple detection strategy that uses magnetic nanoparticles (MNPs) and a novel MagR-MazE fusion protein for molecular diagnostics to facilitate sensitive detection. This study has engineered a novel MNP conjugate that can be generated easily, without using many chemical reagents. The technique is a nucleic acid detection method, using MagR-MazE fusion protein-conjugated MNPs, where the results can be visualized with the naked eye, regardless of the oligonucleotide sequences of the target in the lateral flow assay. This method could sensitively detect polymerase chain reaction (PCR) products of 16S ribosomal RNA (rRNA) and the 2019-nCoV-N-positive control gene in 5 min. It shows a low limit of detection (LoD) of 0.013 ng/µL for dsDNA. It is simpler and more rapid, sensitive, and versatile than other techniques, making it suitable for point-of-care testing. The proposed detection system and MNP conjugation strategy using a fusion protein can be widely applied to various fields requiring rapid on-site diagnosis.