Molecular Diagnostic Applications Research Articles

A Fusion of Taq DNA Polymerase with the CL7 Protein from Escherichia coli Remarkably Improves DNA Amplification.

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

Lateral Flow Biosensor for On-Site Multiplex Detection of Viruses Based on One-Step Reverse Transcription and Strand Displacement Amplification.

Respiratory pathogens pose a huge threat to public health, especially the highly mutant RNA viruses. Therefore, reliable, on-site, rapid diagnosis of such pathogens is an urgent need. Traditional assays such as nucleic acid amplification tests (NAATs) have good sensitivity and specificity, but these assays require complex sample pre-treatment and a long test time. Herein, we present an on-site biosensor for rapid and multiplex detection of RNA pathogens. Samples with viruses are first lysed in a lysis buffer containing carrier RNA to release the target RNAs. Then, the lysate is used for amplification by one-step reverse transcription and single-direction isothermal strand displacement amplification (SDA). The yield single-strand DNAs (ssDNAs) are visually detected by a lateral flow biosensor. With a secondary signal amplification system, as low as 20 copies/μL of virus can be detected in this study. This assay avoids the process of nucleic acid purification, making it equipment-independent and easier to operate, so it is more suitable for on-site molecular diagnostic applications.

A Simple and Robust Exponential Amplification Reaction (EXPAR)-Based Hairpin Template (exp-Hairpin) for Highly Specific, Sensitive, and Universal MicroRNA Detection.

Specific and sensitive detection of microRNAs continues to encounter significant challenges, especially in the development of rapid and efficient isothermal amplification strategies for point-of-care settings. The exponential amplification reaction (EXPAR) has garnered significant attention owing to its simplicity and rapid amplification of signals within a short period. However, a substantial loss of amplification efficiency, difficulty in distinguishing closely related homologous sequences, and adapting the designed templates to other targets seriously hamper the practical application of the EXPAR. In this work, a hairpin template tailored for the EXPAR system (exp-Hairpin) was constructed by adding identical trigger sequences and enzyme cleavage sites on two arms of the hairpin, achieving theoretically more than 2n amplification efficiency and minimal background amplification of EXPAR. Modulating the stability of the exp-Hairpin template by increasing the stem length, the specificity of detecting target miRNA in highly homologous sequences could be significantly improved. Using miRNA let-7a as a target model, the exp-Hairpin with 8 bp stem length for EXPAR amplification curves could effectively distinguish target let-7a and nontarget let-7b/7c/7f/7g/7i homologous sequences. This strategy enabled the sensitive and accurate analysis of let-7a in diluted human serum with satisfactory recoveries. By simply replacing the loop recognition sequence of exp-Hairpin, the specific detection of miR-200b was also achieved, demonstrating the universality of this strategy. The exp-Hairpin EXPAR accelerates simple and rapid molecular diagnostic applications for short nucleic acids.

Clinical application of molecular diagnostics in myeloid disorders: recommendations for implementing a common strategy in Spanish centers

Clinical application of molecular diagnostics in myeloid disorders: recommendations for implementing a common strategy in Spanish centers

Fully Integrated Pneumatic-Free and Magnet-Free CMOS Ferrofluidic Platform for Comprehensive Biomolecular Processing.

This article presents a fully integrated CMOS ferrofluidic platform featuring on-chip three-electrode electrochemical cells, temperature regulators, and magnetic sensors. The proposed platform consists of 25 ferrofluidic pixels and 2 magnetic sensors. Each ferrofluidic pixel comprises a spiral inductor, a three-electrode electrochemical cell, a temperature sensor, and a localized Joule heater. Unlike pneumatic-based platforms, this ferrofluidic platform does not require an external pneumatic pump to drive droplets. Instead, the on-chip spiral inductors generate magnetic fields to manipulate the ferrofluidic droplets. Additionally, these inductors are repurposed as heat radiators. The CMOS ferrofluidic platform is implemented using a 45-nm CMOS SOI process. Theoretical analyses of ferrofluidic control and magnetic sensing are conducted to understand the relationship between ferrofluidic movement conditions and the integrated magnetic sensor. The on-chip electrochemical potentiostat is characterized using various concentrations of methylene blue solution, and the variation in the electrochemical sensor is measured. As proof of concept, biological measurements with on-chip real-time recombinase polymerase amplification (RT-RPA) are demonstrated. The proposed platform offers a fully integrated solution for ferrofluidic manipulation, sensing, and temperature regulation without the need for external bulky equipment, thereby supporting advanced biomolecular processing. While RT-RPA is used here solely for demonstration purposes, our ferrofluidic multi-functional CMOS array platform is also capable of processing a wide range of other molecular analytes. This versatility underscores the platform's potential for broad applications in molecular diagnostics and bioanalytical research.

Generic strategy for the synthesis of highly specific Au/MIP nanozymes and their application in homogeneous assays

Enzymes are highly complex nanomachines produced by cells with the ability to catalyse diverse chemical reactions. Their applications in biotechnology and molecular diagnostics are widespread. However, most enzymes suffer from...

A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses

DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract total bacterial DNA from environmental samples based on the sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in as little as 10 min at room temperature with dilution, or the DNA can be further purified using silica-coated paramagnetic beads. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrated that an impinger and portable pump can efficiently capture bacteria from the air in poultry facilities for rapid NaOH extraction to quantify total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well suited for routine, high-throughput screening and for metagenomic analyses for specific pathogens, even in resource-limited situations.

Picomolar-Level Sensing of Cannabidiol by Metal Nanoparticles Functionalized with Chemically Induced Dimerization Binders.

Simple and fast detection of small molecules is critical for health and environmental monitoring. Methods for chemical detection often use mass spectrometers or enzymes; the former relies on expensive equipment, and the latter is limited to those that can act as enzyme substrates. Affinity reagents like antibodies can target a variety of small-molecule analytes, but the detection requires the successful design of chemically conjugated targets or analogs for competitive binding assays. Here, we developed a generalizable method for the highly sensitive and specific in-solution detection of small molecules, using cannabidiol (CBD) as an example. Our sensing platform uses gold nanoparticles (AuNPs) functionalized with a pair of chemically induced dimerization (CID) nanobody binders (nanobinders), where CID triggers AuNP aggregation and sedimentation in the presence of CBD. Despite moderate binding affinities of the two nanobinders to CBD (equilibrium dissociation constants KD of ∼6 and ∼56 μM), a scheme consisting of CBD-AuNP preanalytical incubation, centrifugation, and electronic detection (ICED) was devised to demonstrate a high sensitivity (limit of detection of ∼100 picomolar) in urine and saliva, a relatively short sensing time (∼2 h), a large dynamic range (5 logs), and a sufficiently high specificity to differentiate CBD from its analog, tetrahydrocannabinol. The high sensing performance was achieved with the multivalency of AuNP sensing, the ICED scheme that increases analyte concentrations in a small assay volume, and a portable electronic detector. This sensing system is readily applicable for wide molecular diagnostic applications.

UK clinicians' attitudes towards the application of molecular diagnostics to guide antibiotic use in ICU patients with pneumonias: a quantitative study.

Molecular diagnostic tests may improve antibiotic prescribing by enabling earlier tailoring of antimicrobial therapy. However, clinicians' trust and acceptance of these tests will determine their application in practice. To examine ICU prescribers' views on the application of molecular diagnostics in patients with suspected hospital-acquired and ventilator-associated pneumonia (HAP/VAP). Sixty-three ICU clinicians from five UK hospitals completed a cross-sectional questionnaire between May 2020 and July 2020 assessing attitudes towards using molecular diagnostics to inform initial agent choice and to help stop broad-spectrum antibiotics early. Attitudes towards using molecular diagnostics to inform initial treatment choices and to stop broad-spectrum antibiotics early were nuanced. Most (83%) were positive about molecular diagnostics, agreeing that using results to inform broad-spectrum antibiotic prescribing is good practice. However, many (58%) believed sick patients are often too unstable to risk stopping broad-spectrum antibiotics based on a negative result. Positive attitudes towards the application of molecular diagnostics to improve antibiotic stewardship were juxtapositioned against the perceived need to initiate and maintain broad-spectrum antibiotics to protect unstable patients.

Precise determination of reaction conditions for accurate quantification in digital PCR by real-time fluorescence monitoring within microwells

Real-time digital polymerase chain reaction (qdPCR) provides enhanced precision in the field of molecular diagnostics by integrating absolute quantification with process information. However, the optimal reaction conditions are traditionally determined through multiple iterative of experiments. Therefore, we proposed a novel approach to precisely determine the optimal reaction conditions for qdPCR using a standard process, employing real-time fluorescence monitoring within microwells. The temperature-sensitive fluorophore intensity presented the real temperature of each microwell. This enabled us to determine the optimal denaturation and annealing time for qdPCR based on the corresponding critical temperatures derived from the melting curves and amplification efficiency, respectively. To confirm this method, we developed an ultrathin laminated chip (UTL chip) and chose a target that need to be absolutely quantitative. The UTL chip was designed using a fluid‒solid‒thermal coupling simulation model and exhibited a faster thermal response than a commercial dPCR chip. By leveraging our precise determination of reaction conditions and utilizing the UTL chip, 40 cycles of amplification were achieved within 18 min. This was accomplished by precisely controlling the denaturation temperature at 2 s and the annealing temperature at 10 s. Furthermore, the absolutely quantitative of DNA showed good correlation (R2 > 0.999) with the concentration gradient detection using the optimal reaction conditions with the UTL chip for qdPCR. Our proposed method can significantly improve the accuracy and efficiency of determining qdPCR conditions, which holds great promise for application in molecular diagnostics.

Low background self-primer EXPAR coupled with colorimetric and lateral flow assay for rapid and sensitive point-of-care detection of miRNA

MicroRNAs (miRNAs) have emerged as powerful biomarkers for various diseases diagnosis, yet their clinical application is greatly plagued by the lack of reliable and rapid detection methods. Here, a simple, fast, and accurate point-of-care testing (POCT) platform for miRNA-155 detection was developed by interfacing self-primer exponential amplification reaction (SPEXPAR) with colorimetric and POCT lateral flow assay (LFA). In this platform, a hairpin template was designed for exponential amplification reaction, which could effectively avoid nonspecific template interactions and eventually effectively avoid undesirable background amplification; the colorimetric assay is based on the SPEXPAR amplicons induced gold nanoparticles (AuNPs) color change; and the LFA detection principle involves the amplicons hybridization with designed probes. Under the optimized conditions, the colorimetric assay could detect miRNA-155 as low as 0.41 pM in less than 35 min; and LFA could detect as low as 100 pM in less than 40 min. Furthermore, the method was adopted to detect miRNA-155 in human serum samples with satisfactory recoveries. In summary, this study offered a promising analytical tool for molecular diagnostic testing application.

Removal of N-Terminal Peptide Impacts Structural Aspects of an IgE-Reactive Recombinant Der p 5

(1) Background: Modification of the structural elements of allergens is widely used in the field of allergies. The goal of the present research was to express, purify, and characterize the shortened recombinant group 5 allergen of Dermatophagoides pteronyssinus (rDer p 5). (2) Methods: rDer p 5 storage stability and aggregation capacity were explored through in silico analysis, dynamic light scattering (DLS), and SDS-PAGE. Serum IgE reactivity and cytokine amount were investigated in sera or cell culture supernatants through ELISA, MULTIPLEX®, and Western blot analysis using sera from sensitized humans from Brazil, Colombia, and Ecuador. (3) Results: Dimeric rDer p 5 was detected through native PAGE, and this result was confirmed by data from DLS. The protein was thermically stable, as it did not degrade at 4 °C for 21 days. The shortened rDer p 5 was classified as a major IgE allergen in Brazil and Colombia, but minor in Ecuador. IL-13, IL-10, IL-1β, and IL-6 were significantly elevated in the sera of rDer p 5-reactive patients. The same cytokines plus IL-5 were more secreted by human cells upon rDer p 5 stimulation. (4) Conclusions: N-terminal peptide deletion led to a higher rDer p 5 folding stability, which, even though dimeric, was an IgE-reactive protein. Therefore, rDer p 5 could be used for molecular diagnostic applications or as backbone for hypoallergen design.

Ultrasensitive electrochemical detection of amyloid-beta oligomers using double amplification strategy by MXene substrate and covalent organic framework-based probe

Most of the existing electrochemical systems failed to achieve satisfactory results in early diagnosis of Alzheimer's disease (AD) owing to a deficiency of effective signal transduction. A new method for the electrochemical detection of AD biomarkers (amyloid-beta oligomers, Aβ1-42 oligomers) was developed based on a double amplification strategy. Titanium carbide (Ti3C2) MXene decorated by gold nanoparticles (Au-MXene) as the electrode substrate not only gave rise to the electrochemical response due to its paradoxical surface area and conspicuous charge mobility, but also provided vast numbers of binding sites for aptamers (Apt) of Aβ1-42 oligomers. Meanwhile, AuNPs were incorporated into covalent organic frameworks (COFs), which were further modified by Apt and electron mediator (toluidine blue, TB). The Apt/TB-Au@COFs composite was utilized as a label because of their improvement of the electron-hole separation efficiency and optimization of the charge-carrier utilization. The proposed electrochemical assay established highly efficient platform for the detection of Aβ1-42 oligomers with a linear range from 0.01 pg mL−1 to 180 pg mL−1 and an ultralow detection limit of 4.27 fg mL−1 (S/N = 3). This biosensing platform had potential applications in molecular diagnostics of AD serum samples.

Performance and Hypothetical Impact on Joint Infection Management of the BioFire Joint Infection Panel: a Retrospective Analysis.

Pathogen identification is key in septic arthritis. Culture-based techniques are challenging, especially when patients have been pretreated with antibiotics or when difficult-to-culture bacteria are encountered. The BioFire joint infection assay (BJA) is a multiplex PCR panel which detects 31 of the most prevalent bacterial and fungal pathogens causing septic arthritis. Here, 123 cryoconserved contemporary synovial fluid samples from 120 patients underwent BJA analysis. Results were compared to those of culture-based diagnostics (standard of care [SOC]). Clinical data were collected, and the possible impact of the molecular diagnostic application on patient management was evaluated. Fifteen of 123 synovial fluid cultures grew bacterial pathogens. All on-panel pathogens (9/15) were correctly identified by the BJA. The BJA identified four additional bacterial pathogens in four SOC-negative cases. BJA sensitivity and specificity were 100% (95% confidence interval [CI], 69.2% to 100%) and 100% (95% CI, 96.8% to 100%), respectively. Compared to the SOC, the BJA would have resulted in faster provision of species identification and molecular susceptibility data by 49 h and 99 h, respectively. Clinical data analysis indicates that in BJA-positive cases, faster species ID could have led to timelier optimization of antibiotic therapy. This retrospective study demonstrates high sensitivity and specificity of the BJA to detect on-panel organisms in bacterial arthritis. The usefulness of the BJA in prosthetic-joint infections is limited, as important pathogens (i.e., coagulase negative staphylococci and Cutibacterium acnes) are not covered. Evidence from patient data analysis suggests that the assay might prove valuable for optimizing patient management in acute arthritis related to fastidious organisms or for patients who received antibiotics prior to specimen collection.

A clinically applicable molecular classification of oncocytic cell thyroid nodules.

Whole-chromosome instability with near-whole genome haploidization (GH) and subsequent endoreduplication is considered a main genomic driver in the tumorigenesis of oncocytic cell thyroid neoplasms (OCN). These copy number alterations (CNA) occur less frequently in oncocytic thyroid adenoma (OA) than in oncocytic carcinoma (OCA), suggesting a continuous process. The current study described the CNA patterns in a cohort of 30 benign and malignant OCN, observed using a next generation sequencing (NGS) panel that assesses genome-wide loss of heterozygosity (LOH) and chromosomal imbalances using 1,500 single nucleotide polymorphisms (SNP) across all autosomes and the X chromosome in DNA derived from cytological and histological samples. Observed CNA patterns were verified using multiparameter DNA flow cytometry with or without whole-genome SNP array analysis and lesser-allele intensity-ratio (LAIR) analysis. On CNA-LOH analysis using the NGS panel, GH type CNA were observed 4 of 11 (36%) OA and 14 of 16 OCA (88%). Endoreduplication was suspected in 8 of 16 (50%) OCA, all with more extensive GH type CNA (p<0.001). Reciprocal chromosomal imbalance type CNA, characterized by (imbalanced) chromosomal copy number gains and associated with benign disease, were observed in 6 of 11 (55%) OA and one equivocal case of OCA. CNA patterns were different between the histopathological subgroups (p<0.001). By applying the structured interpretation and considerations provided by the current study, CNA-LOH analysis using an NGS panel that is feasible for daily practice may be of great added value to the widespread application of molecular diagnostics in the diagnosis and risk stratification of OCN.

Highly purified DNA-containing cell envelopes from fungi for direct use in PCR

Efficient DNA sample preparation from fungi with the rigid cell walls is still critical for successful polymerase chain reaction (PCR), one of the basic platforms in molecular diagnostics of fungi, especially in medical mycology. Common methods that involve different chaotropes to yield DNA samples have found a limited application for fungi. Here we describe a novel procedure for efficient production of permeable fungal cell envelopes with DNA inside as suitable templates for PCR. This procedure is facile, relies on boiling of fungal cells in aqueous solutions of selected chaotropic agents and additives and enables to remove RNA and proteins from PCR template samples. The use of chaotropic solutions containing 7 M urea, 1% sodium dodecyl sulfate (SDS), up to100 mM ammonia and/or 25 mM sodium citrate was the best option to yield highly purified DNA-containing cell envelopes from all fungal strains under study, including clinical Candida and Cryptococcusisolates. After treatment with the selected chaotropic mixtures, the fungal cell walls had undergone loosening and were no longer a barrier to release DNA in PCR as evident from electron microscopy examinations and successful target gene amplifications. Overall, the developed simple, fast, and low-cost approach to produce PCR-suitable templates in the form of DNA encased by permeable cell walls can find application in molecular diagnostics.

Unlocking the power of precision medicine for pediatric low-grade gliomas: molecular characterization for targeted therapies with enhanced safety and efficacy.

In the past decade significant advancements have been made in the discovery of targetable lesions in pediatric low-grade gliomas (pLGGs). These tumors account for 30-50% of all pediatric brain tumors with generally a favorable prognosis. The latest 2021 WHO classification of pLGGs places a strong emphasis on molecular characterization for significant implications on prognosis, diagnosis, management, and the potential target treatment. With the technological advances and new applications in molecular diagnostics, the molecular characterization of pLGGs has revealed that tumors that appear similar under a microscope can have different genetic and molecular characteristics. Therefore, the new classification system divides pLGGs into several distinct subtypes based on these characteristics, enabling a more accurate strategy for diagnosis and personalized therapy based on the specific genetic and molecular abnormalities present in each tumor. This approach holds great promise for improving outcomes for patients with pLGGs, highlighting the importance of the recent breakthroughs in the discovery of targetable lesions.

Novel Bifunctional Affibody Molecules with Specific Binding to Both EBV LMP1 and LMP2 for Targeted Therapy of Nasopharyngeal Carcinoma.

Antibodies are considered highly specific therapeutic agents in cancer medicines, and numerous formats have been developed. Among them, bispecific antibodies (BsAbs) have gained a lot of attention as a next-generation strategy for cancer therapy. However, poor tumor penetration is a major challenge because of their large size and thus contributes to suboptimal responses within cancer cells. On the other hand, affibody molecules are a new class of engineered affinity proteins and have achieved several promising results with their applications in molecular imaging diagnostics and targeted tumor therapy. In this study, an alternative format for bispecific molecules was constructed and investigated, named ZLMP110-277 and ZLMP277-110, that targets Epstein-Barr virus latent membrane protein 1 (LMP1) and latent membrane protein 2 (LMP2). Surface plasmon resonance (SPR), indirect immunofluorescence assay, co-immunoprecipitation, and near-infrared (NIR) imaging clearly demonstrated that ZLMP110-277 and ZLMP277-110 have good binding affinity and specificity for both LMP1 and LMP2 in vitro and in vivo. Moreover, ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, significantly reduced the cell viability of C666-1 and CNE-2Z as compared to their monospecific counterparts. ZLMP110-277 and ZLMP277-110 could inhibit phosphorylation of proteins modulated by the MEK/ERK/p90RSK signaling pathway, ultimately leading to suppression of oncogene nuclear translocations. Furthermore, ZLMP110-277 and ZLMP277-110 showed significant antitumor efficacy in nasopharyngeal carcinoma-bearing nude mice. Overall, our results demonstrated that ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, are promising novel prognostic indicators for molecular imaging and targeted tumor therapy of EBV-associated nasopharyngeal carcinoma.

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR.

Polymerase chain reaction (PCR) has extensive bioanalytical applications in molecular diagnostics and genomic research studies for rapid detection and precise genomic amplification. Routine integrations for analytical workflow indicate certain limitations, including low specificity, efficiency, and sensitivity in conventional PCR, particularly towards amplifying high guanine-cytosine (GC) content. Further, there are many ways to enhance the reaction, for example, using different PCR strategies such as hot-start/touchdown PCR or adding some special modifications or additives such as organic solvents or compatible solutes, which can improve PCR yield. Due to the widespread use of bismuth-based materials in biomedicine, which have not yet been used for PCR optimization, this attracts our attention. In this study, two bismuth-based materials that are inexpensive and readily available were used to optimize GC-rich PCR. The results demonstrated that ammonium bismuth citrate and bismuth subcarbonate effectively enhanced PCR amplification of the GNAS1 promoter region (∼84% GC) and APOE (75.5% GC) gene of Homo sapiens mediated by Ex Taq DNA polymerase within the appropriate concentration range. Combining DMSO and glycerol additives was critical in obtaining the target amplicons. Thus, the solvents mixed with 3% DMSO and 5% glycerol were used in bismuth-based materials. That allowed for better dispersion of bismuth subcarbonate. As for the enhanced mechanisms, the surface interaction of PCR components, including Taq polymerase, primer, and products with bismuth-based materials, was maybe the main reason. The addition of materials can reduce the melting temperature (Tm), adsorb polymerase and modulate the amount of active polymerase in PCR, facilize the dissociation of DNA products, and enhance the specificity and efficiency of PCR. This work provided a class of candidate enhancers for PCR, deepened our understanding of the enhancement mechanisms of PCR, and also explored a new application field for bismuth-based materials.

Copy number variation (CNV) calling of MET, MYC, and ERBB2 using PiVAT bioinformatic platform.

e15145 Background: CNV in tumor-associated genes such as MET, MYC and ERBB2 amplification can lead to cancer progression and poor survival in multiple cancers, and is associated with treatment regimen decision. Whole genome sequencing (WGS) and comprehensive genomic profiling (CGP) panels covering hundreds of genes are currently used to detect CNV in hospitals and CLIA-accredited laboratories. However, cost and complex workflow of WGS/CGP restrict their application in clinical molecular diagnostics. CGP panel covering dozens of genes based on targeted amplicon sequencing (TAS) method is preferred, but it is not ideal to detect CNV accurately due to unique methodology. Here we established a CNV calling toolkit in PiVAT bioinformatic platform to solve CNV calling problems in small-size TAS panel. Methods: 46 CNV positive (CNV+) and 14 CNV negative (CNV-) samples were sequenced using MGISEQ-2000 with a paired end, 2x100 read length protocol and adapter sequences were trimmed from 3’ ends of each read. PiVAT’s CNV calling method is based on double coverage normalization, including one per-sample normalization and one per-amplicon normalization. CNV- samples were used as normalization references. MET, MYC and ERBB2 copy number was investigated to determine sensitivity, specificity, precision, and accuracy of the overall test. Copy number deletion is defined as ratio &lt; 0.8 and amplification is defined as ratio &gt; 1.2. PiVAT CNV output calls were compared against excepted copy number to calculate and plot the concordance. CNVkit and Control-FREEC, 2 other CNV calling tools, were chosen as comparators to compare ERBB2, MET and MYC copy number levels with PiVAT’s CNV caller. Results: Sequencing results from all 46 CNV+ samples showed high coverage uniformity, with &gt; 90% of sites having base coverage depth &gt; 20% of mean coverage for all samples. Average mapping rate and on-target rate of sequencing run, analyzed by PiVAT, were 98.98±0.41% and 98.39±0.45%. Results from PiVAT showed a sensitivity of 97.83% and a specificity of 100% for MET, MYC, ERBB2 amplifications. PiVAT estimated copy number more accurately compared with CNVkit and Control-FREEC. PiVAT’s R2, measuring degree of difference from expected copy number, was greater than CNVkit and Control-FREEC for MET, MYC and ERBB2, with values at 0.78, 0.69 and 0.96, respectively. In contrast, CNVkit and Control-FREEC were on average 77.38% and 164.04% worse. For CNVkit and Control-FREEC, many of MET, MYC and ERBB2 CNV calls were seen at a much lower magnitude than expected. Conclusions: Our studies have shown that PiVAT is a reliable tool for calling CNVs. PiVAT’s sensitivity and accuracy were calculated to be &gt; 97%. And PiVAT outperformed other CNV calling tools, CNVkit and Control-FREEC. This demonstrates that PiVAT’s CNV caller can be used to provide CNV results from TAS panel, and guide relevant cancer diagnosis and clinical decision more accurately.