Method For Detection Of Mutations Research Articles

Diagnostic Value of Conventional Polymerase Chain Reaction for Detecting BRAF V595E Mutation in Liquid and Tissue Specimens of Canine Urothelial and Prostate Carcinomas.

Canine urothelial carcinoma (UC) and prostatic carcinoma (PC) often present diagnostic challenges due to their anatomical locations. The BRAF V595E mutation, analogous to the human BRAF V600E mutation, has been identified in UC and PC. Digital PCR of urine is a non-invasive diagnostic method of mutation detection, but the availability of the necessary equipment is limited. This study aimed to develop a conventional PCR to detect the BRAF V595E mutation in urine and prostatic wash specimens from dogs with UC or PC. Specific primers for detecting wild-type and mutant BRAF V595E genes were validated in 34 formalin-fixed paraffin-embedded (FFPE) tissues, 116 urine samples, and 9 prostatic wash specimens. The results showed that the BRAF V595E mutation detection rate for UC and PC in the tissues was 51.6%. The detection rate in liquid specimens from dogs with lower urinary tract or prostate masses was 53.2%. Of the 41 cases with follow-up, 16 were further diagnosed with UC or PC, with 75% of liquid specimens from these dogs showing the BRAF V595E mutation. This conventional PCR method provides a reliable and non-invasive screening tool for UC and PC in dogs, especially in settings without advanced equipment.

Neomorphic IDH Heterodimer Mutants: An Underestimated Point in Cancer Pathogenesis.

Mutations in an essential metabolic enzyme, isocitrate dehydrogenase (IDH), were found in many cancers. The impact of IDH1 and IDH2 proteoforms mutations can vary and de-pend on the cancer type and other genetic alterations. The wild-type IDH1/2 consists of two identical subunits, but the mutant enzyme is a heterodimer of mutant and wild-type subunits, while the mutant homodimer loses its catalytic activity. Thus, the balance of expression of wild-type and mutant proteoforms directly affects enzyme neomorphic activity, cell metabolic por-trait, and, therefore, cell survival and proliferation rates. Here, we generalize the influence of the presence of IDH mutations and the expression of mutant and wild-type proteoforms for various nosologies to demonstrate the deficiency in knowledge about the mutual distribution of the pro-teoforms in cancer cells. The review is supplemented with a brief description of IDH mutations' role in cell metabolic reprogramming and a summary of methods for IDH mutation detection. Eventually, we highlight the necessity of assessing the expression of wild-type and mutated IDH quantitatively, which can help create and deliver personalized approaches for tumor therapy.

Highly Sensitive 3D‐Nanoplasmonic‐Based Epidermal Growth Factor Receptor Mutation Multiplex Assay Chip for Liquid Biopsy

Economical mutation detection method with high analytical and clinical sensitivity is necessary for early cancer diagnosis and screening. In this study, a novel 3D‐nanoplasmonic‐based multiplex mutation assay chip is developed to detect epidermal growth factor receptor (EGFR) mutations. This assay kit comprises a 3D‐nanoplasmonic substrate immobilized with capture probes and primer–probe sets for recombinase polymerase amplification, wild‐type inhibition, and fluorescence detection, enabling multiplex detection of EGFR exon 19 deletions, exon 20 insertions, and exon 21 L858R point mutations. The strategy facilitates the detection of all deletions and insertions within the target region with extremely high analytical sensitivity, detecting as low as 1 × 10−9% mutation frequency, implying three copies/reactions and 100 zM. The synergistic effects of plasmon‐enhanced fluorescence from the 3D‐nanoplasmonic substrate and wild‐type inhibitor contribute to this high analytical sensitivity. Moreover, the developed chip exhibits 100% accuracy in the clinical testing of plasma samples from normal individuals and patients with benign lung tumor and malignant lung tumor. With high sensitivity and multiplexing capabilities, this assay operates at a low reaction temperature (around 37 °C) and requires a short processing time, ≈70 min post‐cell‐free DNA extraction. These features make the chip a valuable tool for easy and widespread cancer screening.

Development of a hydrogen peroxide fluorescent probe for the rapid discrimination of IDH1 mutant gliomas from the wild-type

Isocitrate dehydrogenase (IDH) mutations are common in gliomas, and recent studies have suggested that these mutants have distinct microenvironments characterized by higher levels of reactive oxygen species (ROS) and a higher pH. The identification of IDH mutants is important for the diagnosis, individualized treatment, and clinical prognosis of gliomas. Sanger sequencing and immunohistochemistry are the standard methods for IDH mutation detection. As the mutations have several types, the detection of multiple gene sites and proteins are required, which means high cost and tedious operations. The high levels of ROS observed in mutIDH gliomas have not been used for mutIDH detection. Fluorescent probe-based detection techniques have emerged as a prevalent method to detect diverse analytes in living systems due to their distinctive benefits of simple operation, high sensitivity and selectivity, fast response, and real-time applicability. In the present study, we report the rapid discrimination of mutIDH1 from wild-type gliomas using the novel fluorescent probe BOD-G that varies in its fluorescence response to different levels of H2O2. BOD-G exhibits high sensitivity and selectivity in its turn-on response to H2O2, and its performance improves as the pH increases. The probe exhibits a much stronger fluorescence in IDH1 mutant U87 cells than in wild-type U87MG cells after 5 min. Importantly, BOD-G displays a bright fluorescence when exposed to fresh mutIDH1 human glioma specimens after 15 min but not with wild-type samples. Thus, BOD-G can be used to discriminate mutIDH1 from wild-type human glioma specimens rapidly by simple fluorescent imaging.

Identification of glucose-6-phosphate dehydrogenase variants by utilizing polymerase chain reaction – Restriction fragment length polymorphism based method

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a widespread genetically inherited enzyme disorder caused by mutation in the G6PD gene located in X chromosome. Although sequencing of the gene is considered as the standard method for detection of mutations, but the process is expensive, laborious, time-consuming and not possible to perform in low resource settings. Thus, the present work was aimed to identify the G6PD variants using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) based method among the ethnic groups of malaria endemic Northeastern part of India. STANDARD G6PD Analyzer was utilized for screening to identify the G6PD deficient malarial patients. Molecular analysis of the deficient samples was done using PCR-RFLP technique. A total of 17 variants were explored using different restriction endonucleases. Five variants viz., Orissa (63.8 %), Kalyan-Kerala/Jamnagar/Rohini (9.02 %), Mediterranean (8.3 %), A+ (11.8 %) and Mahidol (1.38 %) were detected among the patients. G6PD Orissa was the major variant among the study population. In malaria endemic regions like Northeast India, a mass screening program for identification of G6PD variants needs to be adopted.

Abstract 320: Development and automation of a streamlined targeted enrichment method for cancer mutation detection

Abstract Next Generation Sequencing (NGS) of DNA is a powerful tool for the detection of genetic variations, including single nucleotide polymorphisms, copy number variation, and small insertions/deletions. Target enrichment technologies enhance DNA sequencing by enabling users to analyze specific regions of interest only, which helps to effectively increase sequencing depth and sample throughput while minimizing cost. However, many targeted DNA library construction workflows are not optimal for routine genetic testing needs due to inconsistent, lengthy workflow. This poses significant challenge to application like cancer mutation profiling that requires detection sensitivity down to 1% or below due to heterogeneous nature of tumor tissue. In addition, high throughput testing with minimal hands on time is desirable with the rapid advance of precision medicine. Magnetic bead separations are commonly used between the individual reactions of targeted DNA library preparation workflows for cleanup and size selection. These bead clean up reactions are not only labor-intensive and tedious procedures but can also greatly contribute to sample-to sample variation especially when employing automated library preparation procedures. With the sophisticated QIAseq Targeted DNA Pro, we developed a novel streamlined targeted DNA workflow that greatly reduces handling variation by replacing the majority of bead clean up steps with enzymatic reactions, which now can be finished in as little as six hours. In combination with the newly developed QIAseq Normalizer kit, the protocol has been successfully implemented on the Hamilton NGS Star liquid handling platform resulting in up to 96 highly uniform, complex and ready-to-sequence libraries that can be effortless generated in one run. This complete automated solution saves researcher up to four hours of hands-on time. When using commercial reference samples, cancer mutations down to 1% were consistently detected with this automated workflow. The presented data demonstrate that this integrated and streamlined workflow does not only provide an automation friendly workflow but also allows to reliably identify low frequent cancer mutations. The applications presented here are for research use only. Not for use in diagnostic procedures. Citation Format: Zhong Wu, Michelle Baird, Cornelia Mechlen, Peter Hahn, Jonathan M. Shaffer. Development and automation of a streamlined targeted enrichment method for cancer mutation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 320.

PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay: A highly sensitive method for detection of MYD88 L265P mutation.

Agarose gel-based conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays are currently used for sensitive detection and quantification of MYD88 L265P mutation. Visual inspection of an agarose gel can often be ambiguous. We propose a new allele-specific quantification PCR (AS-qPCR) assay, PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay, that uses Intercalating Nucleic Acid (INA®) technology for increased affinity and specificity. This study compares PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay with conventional AS-PCR. We included a total of 102 peripheral and bone marrow blood samples from 94 patients with a lymphoproliferative disorder. Droplet digital PCR (ddPCR) was used as a third method in case of discrepancy. A positive percent agreement of 100% (95% CI 0.92-1.0) and a negative percent agreement of 98% (95% CI 0.90-1.0) were found between the conventional AS-PCR and the AS-qPCR methods. Including the ddPCR results to validate the discrepant cases, the sensitivity and specificity of PlentiPlex™ MYD88 Waldenström lymphoma qPCR Assay were 1.0 (95% CI 0.97-1.0) and 1.0 (95% CI 0.96-1.0), respectively. Our data demonstrate that PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay is a fast, highly sensitive, and specific method for the detection of MYD88 L265P compared with conventional AS-PCR.

Rapid Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Protocol for Detection of Y132F Mutation in Fluconazole Resistant Candida parapsilosis.

There is an emerging fluconazole resistance in Candida parapsilosis in recent years. The leading mechanism causing azole resistance in C. parapsilosis is the Y132F codon alteration in the ERG11 gene which encodes the target enzyme of azole drugs. In this study, we evaluated the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method for rapid detection of the Y132F mutation in fluconazole nonsusceptible C. parapsilosis. Antifungal susceptibility tests for detection of fluconazole resistance were performed by broth microdilution according to the CLSI guidelines. All susceptible and nonsusceptible C. parapsilosis isolates were analyzed for ERG11 mutations with Sanger sequencing. T-ARMS-PCR was fully concordant with the Sanger sequencing (100% of sensitivity and specificity) for detection of Y132F mutations. T-ARMS-PCR method could be a rapid, simple, accurate, and economical assay in the early detection of the most common cause of fluconazole resistance in C. parapsilosis isolates. In routine laboratories with high C. parapsilosis isolation rates, performing the T-ARMS-PCR for early detection of the most common reason of fluconazole resistance in C. parapsilosis, could be a life-saving approach for directing antifungal therapy before obtaining the definitive antifungal susceptibility tests results.

Determination of Helicobacter pylori primary resistance to levofloxacin in gastric mucosal biopsy samples using real-time polymerase chain reaction

Objective. To study the primary resistance of Helicobacter pylori (H. pylori) to levofloxacin in residents of Gomel region by real-time polymerase chain reaction (RT PCR).Materials and methods. The study included 170 patients diagnosed with gastritis and duodenitis, K29, median age - years (25% and 75% 37 and 61 years). According to the questionnaire data of the patients, eradication therapy with levofloxacin was not performed for them. To determine the resistance of H. pylori to levofloxacin we used RT PCR. Results. Out of 170 DNA samples analyzed, 8 samples had doubtful results and according to the methodology for recording the results are subject to rearrangement from the DNA isolation stage. The remaining 162 samples were positive for the β-actin gene (internal control sample ICS) and were taken into account in further analysis (Ct, CY5 19.6-27.4). 16sRNA gene DNA (Ct, ROX 19.5-30.04), indicative of bacterial infection, was confirmed in 152 samples (93.8%). DNA of the gyrA gene (point mutations A259T, T261C, G261A, G271A, G271T and A272G) was detected in 19 of 152 DNA samples, and H. pylori resistance to levofloxacin was 12.5 %, (Ct, Hex 23.2-30.7). The positive control samples had characteristic curve growth on the corresponding detection channels, while the negative samples showed no curve growth.Conclusion. Primary resistance of H. pylori to levofloxacin in residents of Gomel region amounted to 12.5%. Mutations of gyrA gene are the most sensitive marker for predicting successful eradication when using fluoroquinolones, in particular levofloxacin. RT PCR is a reliable method of mutation detection and allows simultaneous detection of H. pylori DNA and resistance to levofloxacin, which significantly reduces the study time.

Comparison of diagnostic methods for detection of BRAFV600E mutation in ameloblastoma.

Ameloblastoma is an aggressively growing, highly recurrent odontogenic jaw tumor. Its association with BRAFV600E mutation is an indication for BRAFV00E-inhibitor therapy The study objective was to identify a sensitive low-cost test for BRAFV600E-positive ameloblastoma. We hypothesized that immunohistochemical staining of formalin-fixed paraffin-embedded tissues for BRAFV600E mutation is a low-cost surrogate for BRAFV600E gene sequencing when laboratory resources are inadequate for molecular testing. Tissues from 40 ameloblastoma samples were retrieved from either formalin-fixed paraffin-embedded blocks, RNAlater™ stabilization solution or samples inadvertently pre-fixed in formalin before transfer to RNAlater™. BRAFV600E mutation was assessed by Direct Sanger sequencing, Amplification Refractory Mutation System-PCR and immunohistochemistry (IHC). BRAFV600E mutation was detected by IHC, Amplification Refractory Mutation System-PCR and Direct Sanger sequencing in 93.33%, 52.5% and 30% of samples respectively. Considering Direct Sanger sequencing as standard BRAFV600E detection method, there was significant difference between the three detection methods (𝜒2 (2) = 31.34, p < 0.0001). Sensitivity and specificity of IHC were 0.8 (95% CI: 0.64-0.90) and 0.9 (95% CI: 0.75-0.99) respectively, while positive predictive value and negative predictive value (NPV) were 0.9 and 0.8 (Fischer's test, p < 0.0001) respectively. Sensitivity and specificity of Amplification Refractory Mutation System-PCR detection method were 0.7 (95% CI: 0.53-0.80) and 0.9 (95% CI = 0.67-0.98) respectively, while PPV and NPV were 0.9 and 0.6 respectively (Fischer's test, p < 0.0001). Low-cost and less vulnerability of IHC to tissue quality make it a viable surrogate test for BRAFV600E detection in ameloblastoma. Sequential dual IHC and molecular testing for BRAFV600E will reduce equivocal results that could exclude some patients from BRAFV600E-inhibitor therapies.

CRISPR-Cas system: A promising tool for rapid detection of SARS-CoV-2 variants.

COVID-19, caused by SARS-CoV-2, remains a global health crisis. The emergence of multiple variants with enhanced characteristics necessitates their detection and monitoring. Genome sequencing, the gold standard, faces implementation challenges due to complexity, cost, and limited throughput. The CRISPR-Cas system offers promising potential for rapid variant detection, with advantages such as speed, sensitivity, specificity, and programmability. This review provides an in-depth examination of the applications of CRISPR-Cas in mutation detection specifically for SARS-CoV-2. It begins by introducing SARS-CoV-2 and existing variant detection platforms. The principles of the CRISPR-Cas system are then clarified, followed by an exploration of three CRISPR-Cas-based mutation detection platforms, which are evaluated from different perspectives. The review discusses strategies for mutation site selection and the utilization of CRISPR-Cas, offering valuable insights for the development of mutation detection methods. Furthermore, a critical analysis of the clinical applications, advantages, disadvantages, challenges, and prospects of the CRISPR-Cas system is provided.

Gene point mutation information translation and detection: Leveraging single base extension and CRISPR/Cas12a

Gene point mutations play a significant role in the development of cancer. Therefore, developing a sensitive, specific, and universally applicable method for detecting gene point mutation is crucial for clinical diagnosis, prognosis, and cancer treatment. Recently, gene point mutation detection methods based on CRISPR/Cas12a detection have emerged. However, existing methods generally lack universality and specificity. In this study, we have developed a CRISPR/Cas12a-based method that combines improved allele-specific polymerase chain reaction and single base extension to translate the point mutation information in the target dsDNA into length information in ssDNA activators to overcome the limitations associated with PAM sequences in the CRISPR/Cas12a system. Our method achieved a detection limit of 0.002% for clinically significant EGFR T790M mutation. The CRISPR/Cas12a system we constructed demonstrates high sensitivity, specificity, and universality in detecting gene point mutations, making it a promising tool for clinical cancer screening.

Prevalence of Factor V Leiden Mutation in Healthy Females of Sindh, Pakistan and Comparison of Three Detection Methods in Resource-Limited Settings

Factor V Leiden (FVL) mutation is related to venous thromboembolism and pregnancy complications. It is highly prevalent among Caucasians; however, few studies have been conducted on the Asian population. Objective: To find the prevalence, genotype, and allelic frequency of FVL mutation in females of Sindh, Pakistan, and to develop a valid and economical method for the detection of FVL mutation in resource-limited settings. Methods: Hundred (n=100), unrelated healthy females of Sindh, Pakistan, were recruited. FVL was detected using three methods, i.e., tetra primer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-sequencing. Prevalence, genotype, and allele frequencies were calculated. We compared the three mutation detection methods to find a suitable protocol for FVL detection in developing countries. Results: The prevalence of FVL mutation was 3% in healthy females of Sindh, Pakistan. No homozygous FVL mutation was found. The results from tetra primer ARMS-PCR, PCR-RFLP, and PCR-sequencing were 100% concordant. Tetra primer ARMS-PCR was a valid, reliable, faster, economical genotyping and screening method. Conclusions: In conclusion, FVL mutation was found in our population, and further studies should be conducted to find its role in thromboembolic and obstetrical complications. In addition, we have suggested tetra primer ARMS-PCR as an appropriate method for FVL detection in resource-limited settings.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

Detection of KatG Mutation in MDR Mycobacterium Tuberculosis Isolates by PCR-RFLP and DNA Sequencing

Objective: Tuberculosis (TB) is among the widespread and rapidly growing infections in the world. Furthermore, TB is one of the major public health problems in Pakistan as every year 48,000 Pakistani dies due to this infection. Pakistan ranks fifth among high burden countries worldwide. As the TB has become most threatening because of the epidemics of human immune deficiency virus (HIV), Covid-19 and the emergence of multi-drug resistance (MDR) strains of Mycobacterium tuberculosis (M. tb). This study was aimed to understand the genetic mechanism of drug resistance in local TB isolates. Methodology: For the genetic studies of INH resistance, KatG (encoding catalase peroxidase) hotspot region was amplified through PCR followed by RFLP and sequencing. Results: The study of PCR-RFLP showed that forty-five out eighty INH resistant M. tb strains had mutations in KatG (codon 315) which is 56.2% of all cases. Sequencing results revealed that this is substitution mutation; AGC to ACC (Ser315Thr). Conclusion: It may be concluded that majority of INH resistance is due to the mutation in the codon 315 of KatG in local isolates. Furthermore, PCR-RFLP technique could be considered as a reliable method for the early detection of KatG mutations in MDR-TB. Bangladesh Journal of Medical Science Vol. 22 No. 04 October’23 Page : 804-808

Biological Microarray for Detection of Somatic Mutations in the Genes of Isocitrate Dehydrogenase 1 and 2

Mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) genes are important diagnostic and prognostic markers in a number of tumors (chondroid tumors, brain tumors, leukemia). To determine mutations in the IDH1/2 genes the method of allele-specific hybridization on a biological microarray was developed, which makes it possible to determine the presence of a mutation in a tumor sample (chondroid tumors and glioblastoma) and the variant of nucleotide substitution. When comparing IDH1/2 mutation detection methods using a biological microarray and DNA melting analysis with a TaqMan probe, full coincidence of results was obtained, with the biochip method making it possible to determine the type of nucleotide substitution without an additional sequencing step.

Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.

Feasibility analysis of rapid gene detection using intraoperative frozen tissues: comparison of intraoperative frozen tissues with paraffin-embedded tissues in epidermal growth factor receptor gene mutation detection of lung adenocarcinoma.

Epidermal growth factor receptor (EGFR) is the driver gene with the highest frequency of mutations in lung adenocarcinoma and can guide the development of targeted therapies. The detection of routine gene mutations must be performed after the preparation of paraffin samples in a standard polymerase chain reaction (PCR) laboratory, which is time-consuming. The Idylla™ EGFR fully automatic PCR system for rapid detection requires no special detection environment and completes the process in only 2.5h. It has been applied to tissues embedded in paraffin. The Idylla™ EGFR automated PCR system was used to detect EGFR gene mutations in intraoperative frozen fresh tissues and paraffin-embedded tissues from 47 enrolled patients with lung adenocarcinoma. The gold standard amplification refractory mutation system (ARMS) method for gene mutation detection was used for verification, and the concordance between the three detection results was compared, to investigate the feasibility of detecting rapid gene mutations in intraoperative frozen samples. The EGFR mutation rate in 47 fresh samples of lung adenocarcinoma was 61.7% (29/47), which is consistent with the mutation level of lung adenocarcinoma in the Asian population (38.8-64.0%). The concordance rate between the Idylla™ frozen tissues and paraffin-embedded tissues was 91.4% (43/47) when compared to the ARMS method, while the coincidence rate between the two methods was 93.6% (44/47). The three methods had a total consistency rate of 89.4% (42/47). The Idylla™ EGFR fully automatic PCR system directly detects EGFR mutations in fresh tissues. The operation is simple, the detection time is short, and the accuracy is high. The detection time is reduced to 1/4-1/3 of the original time while meeting clinical standards for detecting the gene status of patients, thus saving crucial time for individualized and accurate treatment of patients. The method has promising clinical application prospects.

Rapid detection of EGFR mutation in CTCs based on a double spiral microfluidic chip and the real-time RPA method.

Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors and spread into the peripheral bloodstream. Mutation detection in CTCs can reveal vital genetic information about the tumors and can be used for "liquid biopsy" to indicate cancer treatment and targeted medication. However, current methods to measure the mutations in CTCs are based on PCR or DNA sequencing which are cumbersome and time-consuming and require sophisticated equipment. These largely limited their applications especially in areas with poor healthcare infrastructure. Here we report a simple, convenient, and rapid method for mutation detection in CTCs, including an example of a deletion at exon 19 (Del19) of the epidermal growth factor receptor (EGFR). CTCs in the peripheral blood of NSCLC patients were first sorted by a double spiral microfluidic chip with high sorting efficiency and purity. The sorted cells were then lysed by proteinase K, and the E19del mutation was detected via real-time recombinase polymerase amplification (RPA). Combining the advantages of microfluidic sorting and real-time RPA, an accurate mutation determination was realized within 2h without professional operation or complex data interpretation. The method detected as few as 3 cells and 1% target variants under a strongly interfering background, thus, indicating its great potential in the non-invasive diagnosis of E19del mutation for NSCLC patients. The method can be further extended by redesigning the primers and probes to detect other deletion mutations, insertion mutations, and fusion genes. It is expected to be a universal molecular diagnostic tool for real-time assessment of relevant mutations and precise adjustments in the care of oncology patients.

Detection of Ultra-Rare ESR1 Mutations in Primary Breast Cancer Using LNA-Clamp ddPCR.

ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors. These mutations are common in metastatic breast cancer; however, these are rare in primary breast cancer. However, these data have been analyzed mainly in formalin-fixed, paraffin-embedded tissue; thus, rare mutations that may be present in primary breast cancer may be overlooked. In this study, we developed a highly sensitive mutation detection method called locked nucleic acid (LNA)-clamp droplet digital PCR (ddPCR) and validated it. The mutation detection sensitivity was substantiated to 0.003%. Then, we used this method to analyze ESR1 mutations in fresh-frozen (FF) tissues of primary breast cancer. cDNA extracted from the FF tissues of 212 patients with primary breast cancers were measured. Twenty-eight ESR1 mutations were found in twenty-seven (12.7%) patients. Sixteen (7.5%) patients had Y537S mutations and twelve (5.7%) had D538G mutations. Two mutations with a variant allele frequency (VAF) of ≥0.1% and twenty-six mutations with a VAF of <0.1% were found. By using this LNA-clamp ddPCR, this study demonstrated the presence of minor clones with a VAF of <0.1% in primary breast cancer.