Accurate and affordable detection of rifampicin and isoniazid resistance in Tuberculosis sputum specimens by multiplex PCR-multiple probes melting analysis.

BACKGROUND: Escalating cases of multidrug-resistant tuberculosis (MDR-TB) pose a major challenge to global TB control efforts, necessitating innovative diagnostics to empower decentralized detection of gene mutations associated with resistance to rifampicin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis (M. tuberculosis) in resource-constrained settings. METHODS: Combining multiplex fluorescent PCR and Multiple Probes Melting Analysis, we identified mutations in the rpoB, katG, ahpC and inhA genes from sputum specimens. We first constructed a reference plasmid library comprising 40 prevalent mutations in the target genes' resistance determining regions and promoters, serving as positive controls. Our assay utilizes a four-tube asymmetric PCR method with specifically designed molecular beacon probes, enabling simultaneous detection of all 40 mutations. We evaluated the assay's effectiveness using DNA isolated from 50 clinically confirmed M. tuberculosis sputum specimens, comparing our results with those obtained from Sanger sequencing and retrospective validation involving bacteriological culture and phenotypic drug susceptibility testing (pDST). We also included the commercial Xpert MTB/RIF assay for accuracy comparison. RESULTS: Our data demonstrated remarkable sensitivity in detecting resistance to RIF and INH, achieving values of 93.33% and 95.24%, respectively, with a specificity of 100%. The concordance between our assay and pDST was 98.00%. Furthermore, the accuracy of our assay was comparable to both Sanger sequencing and the Xpert assay. Importantly, our assay boasts a 4.2-h turnaround time and costs only $10 per test, making it an optimal choice for peripheral healthcare settings. CONCLUSION: These findings highlight our assay's potential as a promising tool for rapidly, accurately, and affordably detecting MDR-TB.

Silencing LINC00987 ameliorates adriamycin resistance of acute myeloid leukemia via miR-4458/HMGA2 axis.

BACKGROUND: Most patients with acute myeloid leukemia (AML) eventually develop drug resistance, leading to a poor prognosis. Dysregulated long gene non coding RNAs (lincRNAs) have been implicated in chemoresistance in AML. Unfortunately, the effects of lincRNAs which participate in regulating the Adriamycin (ADR) resistance in AML cells remain unclear. Thus, the purpose of this study is to determine LINC00987 function in ADR-resistant AML. METHODS: In this study, ADR-resistant cells were constructed. LINC00987, miRNAs, and HMGA2 mRNA expression were measured by qRT-PCR. P-GP, BCRP, and HMGA2 protein were measured by Western blot. The proliferation was analyzed by MTS and calculated IC50. Soft agar colony formation assay and TUNEL staining were used to analyze cell colony formation and apoptosis. Xenograft tumor experiment was used to analyze the xenograft tumor growth of ADR-resistant AML. RESULTS: We found that higher expression of LINC00987 was observed in AML patients and associated with poor overall survival in AML patients. LINC00987 expression was increased in ADR-resistant AML cells, including ADR/MOLM13 and ADR/HL-60 cells. LINC00987 downregulation reduces ADR resistance in ADR/MOLM13 and ADR/HL-60 cells in vitro and in vivo, while LINC00987 overexpression enhanced ADR resistance in MOLM13 and HL-60 cells. Additionally, LINC00987 functions as a competing endogenous RNA for miR-4458 to affect ADR resistance in ADR/MOLM13 and ADR/HL-60 cells. HMGA2 is a target of miR-4458. LINC00987 knockdown and miR-4458 overexpression reduced HMGA2 expression. HMGA2 overexpression enhanced ADR resistance, which reversed the function of LINC00987 silencing in suppressing ADR resistance of ADR/MOLM13 and ADR/HL-60 cells. CONCLUSIONS: Downregulation of LINC00987 weakens ADR resistance by releasing miR-4458 to deplete HMGA2 in ADR/MOLM13 and ADR/HL-60. Therefore, LINC00987 may act as the therapeutic target for treating chemoresistant AML.

Development of novel parameters for pathogen identification in clinical metagenomic next-generation sequencing.

Introduction: Metagenomic next-generation sequencing (mNGS) has emerged as a powerful tool for rapid pathogen identification in clinical practice. However, the parameters used to interpret mNGS data, such as read count, genus rank, and coverage, lack explicit performance evaluation. In this study, the developed indicators as well as novel parameters were assessed for their performance in bacterium detection. Methods: We developed several relevant parameters, including 10M normalized reads, double-discard reads, Genus Rank Ratio, King Genus Rank Ratio, Genus Rank Ratio*Genus Rank, and King Genus Rank Ratio*Genus Rank. These parameters, together with frequently used read indicators including raw reads, reads per million mapped reads (RPM), transcript per kilobase per million mapped reads (TPM), Genus Rank, and coverage were analyzed for their diagnostic efficiency in bronchoalveolar lavage fluid (BALF), a common source for detecting eight bacterium pathogens: Acinetobacter baumannii, Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Hemophilus influenzae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Aspergillus fumigatus. Results: The results demonstrated that these indicators exhibited good diagnostic efficacy for the eight pathogens. The AUC values of all indicators were almost greater than 0.9, and the corresponding sensitivity and specificity values were almost greater than 0.8, excepted coverage. The negative predictive value of all indicators was greater than 0.9. The results showed that the use of double-discarded reads, Genus Rank Ratio*Genus Rank, and King Genus Rank Ratio*Genus Rank exhibited better diagnostic efficiency than that of raw reads, RPM, TPM, and in Genus Rank. These parameters can serve as a reference for interpreting mNGS data of BALF. Moreover, precision filters integrating our novel parameters were built to detect the eight bacterium pathogens in BALF samples through machine learning. Summary: In this study, we developed a set of novel parameters for pathogen identification in clinical mNGS based on reads and ranking. These parameters were found to be more effective in diagnosing pathogens than traditional approaches. The findings provide valuable insights for improving the interpretation of mNGS reports in clinical settings, specifically in BALF analysis.

A novel design of DNA duplex containing programmable sensing sites for nanopore-based length-resolution reading and applications for Pb2+ and cfDNA analysis.

Glass nanopore is an ideal candidate for biosensors due to its unique advantages such as label-free analysis, single-molecule sensitivity, and easy operation. Previous studies have shown that glass nanopores can distinguish different lengths of double-stranded DNA (dsDNA) at the same time with the length-resolution ability. Based on this, we proposed a novel design of a dsDNA block containing a programmable sensing site inside, which can be programmed to respond to different target molecules and cleaved into two smaller DNA blocks. When programming the sensing site with different sequences, for example, programming it as the substrate of GR-5 DNAzyme and CRISPR-Cas12a system, the DNA block could realize Pb2+ and cfDNA detection with the length-resolution ability of the glass nanopore. This strategy achieved a Pb2+ detection range from 0.5 nM to 100 nM, with a detection limit of 0.4 nM, and a BRCA-1 detection range from 1 pM to 10 pM, with a detection limit of 1 pM. The programable sensing site is easy to design and has strong expandability, which gives full play to the advantages of glass nanopore in length-resolution ability for dsDNA, and is expected to become an optional design for biosensing strategy for the glass nanopore as a biosensing platform.

Clinical diagnostic biomarker "circulating tumor cells" in breast cancer - a meta-analysis.

Objective: Using meta-analysis, we evaluate circulating tumor cells(CTCs) as a potential diagnostic tool for breast cancer. Methods: A document search was conducted using publicly available databases up to May 2021. Specific inclusion and exclusion criteria were formulated and summarize relevant data through literature types, research types, case populations, samples, etc. Subgroup analysis of documents based on regions, enrichment methods, and detection methods. The included research projects were evaluated using DeeKs' bias, and evaluation indicators such as specificity (SPE), sensitivity (SEN), diagnosis odds ratio (DOR) were used as evaluation indicators. Results: 16 studies on the use of circulating tumor cells to diagnose breast cancer were included in our meta-analysis. Overall sensitivity value was 0.50 (95%CI:0.48-0.52), specificity value was 0.93 (95%CI:0.92- 0.95), DOR value was 33.41 (95%CI:12.47-89.51), and AUC value was 0.8129. Conclusion: In meta-regressions and subgroup analysis, potential heterogeneity factors were analyzed, but the source of heterogeneity is still unclear. CTCs, as a novel tumor marker, have a good diagnostic value, but its enrichment and detection methods still need to continue to be developed to improve detection accuracy. Therefore, CTCs can be used as an auxiliary means of early detection, which is helpful to the diagnosis and screening of breast cancer.

Photoelectrochemical immunoassay for thyroglobulin on nanogold-functionalized BiVO4 photoanode coupling with enzymatic biocatalytic precipitation.

Methods derived from photoelectrochemical (PEC) have been constructed for immunoassays, but most involve the split-type immunoreaction modes, and thus easily cause unpredictable intermediate precision. Herein, we innovatively designed an integrated PEC immunosensing platform for the quantitative monitoring of thyroglobulin (TG) on the gold nanoparticles (AuNPs)-functionalized BiVO4 photoanode coupling with enzymatic biocatalytic precipitation (EBCP). This sensing system could simultaneously implement the immunoreaction and photocurrent measurement. Anti-TG capture antibodies were modified onto AuNPs-decorated BiVO4 photoelectrode. A sandwich-type immunoreaction was carried out in the presence of target TG using horseradish peroxidase (HRP)-conjugated anti-TG detection antibody. The carried HRP molecules catalyzed 4-chloro-1-naphthol (4-CN) to generate an insoluble benzo-4-chlorohexadienone product on the photoanode in the presence of peroxide hydrogen, thereby decreasing the photocurrent. Under optimal conditions, the PEC immunosensors gave good photocurrent responses toward target TG within the dynamic range of 0.01-10 ng mL-1 at a detection limit of 7.6 pg mL-1. Good repeatability and precision, high specificity and acceptable storage stability were acquired during the measurement. No significant differences were encountered for screening 15 human serum specimens between the developed PEC immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) method for the detection of target TG. Significantly, PEC immunosensing system offers promise for simple and cost-effective analysis of disease-related biomarkers.

Maternal Western-style diet remodels the transcriptional landscape of fetal hematopoietic stem and progenitor cells in rhesus macaques.

Maternal obesity adversely impacts the in utero metabolic environment, but its effect on fetal hematopoiesis remains incompletely understood. During late development, the fetal bone marrow (FBM) becomes the major site where macrophages and B lymphocytes are produced via differentiation of hematopoietic stem and progenitor cells (HSPCs). Here, we analyzed the transcriptional landscape of FBM HSPCs at single-cell resolution in fetal macaques exposed to a maternal high-fat Western-style diet (WSD) or a low-fat control diet. We demonstrate that maternal WSD induces a proinflammatory response in FBM HSPCs and fetal macrophages. In addition, maternal WSD consumption suppresses the expression of B cell development genes and decreases the frequency of FBM B cells. Finally, maternal WSD leads to poor engraftment of fetal HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2rγ-/- mice. Collectively, these data demonstrate for the first time that maternal WSD impairs fetal HSPC differentiation and function in a translationally relevant nonhuman primate model.

Glucose oxidase-encapsulated liposomes for amplified autofluorescence-free immunoassay of a prostate-specific antigen with photoluminescence of CePO4:Tb nanocrystals.

Lanthanide-doped inorganic nanocrystals have attracted extensive attention due to their long luminescence lifetime and large Stokes shift. In this work, an immunosensing platform based on CePO4:Tb (CPOT) was successfully constructed, which could avoid the autofluorescence interference of complex biological matrices. Specifically, CPOT was synthesized by a solvothermal method, which exhibited H2O2-responsive luminescence behavior. Taking advantage of this feature, an autofluorescence-free immunosensor with CPOT as the probe and H2O2 as the quencher was developed to detect prostate-specific antigen (PSA). Functionalized liposomes were used to encapsulate glucose oxidase (GOD) and labeled on detection antibodies to improve the sensitivity of the probe. Under the proven optimal experimental conditions, the developed autofluorescence-free immunosensor exhibited a linear luminescence response to the logarithm of PSA concentration (0.005-25 ng mL-1) with a limit of detection (LOD) of 3.25 pg mL-1. The performance shows that the autofluorescence-free immunosensor based on this strategy opens up a new field of vision for clinical PSA detection.

Differentiation between glioma recurrence and treatment effects using amide proton transfer imaging: A mini-Bayesian bivariate meta-analysis.

Background: Amide proton transfer (APT) imaging as an emerging MRI approach has been used for distinguishing tumor recurrence (TR) and treatment effects (TEs) in glioma patients, but the initial results from recent studies are different. Aim: The aim of this study is to systematically review and quantify the diagnostic performance of APT in assessing treatment response in patients with post-treatment gliomas. Methods: A systematic search in PubMed, EMBASE, and the Web of Science was performed to retrieve related original studies. For the single and added value of APT imaging in distinguishing TR from TEs, we calculated pooled sensitivity and specificity by using Bayesian bivariate meta-analyses. Results: Six studies were included, five of which reported on single APT imaging parameters and four of which reported on multiparametric MRI combined with APT imaging parameters. For single APT imaging parameters, the pooled sensitivity and specificity were 0.85 (95% CI: 0.75-0.92) and 0.88 (95% CI: 0.74-0.97). For multiparametric MRI including APT, the pooled sensitivity and specificity were 0.92 (95% CI: 0.85-0.97) and 0.83 (95% CI: 0.55-0.97), respectively. In addition, in the three studies reported on both single and added value of APT imaging parameters, the combined imaging parameters further improved diagnostic performance, yielding pooled sensitivity and specificity of 0.91 (95% CI: 0.80-0.97) and 0.92 (95% CI: 0.79-0.98), respectively, but the pooled sensitivity was 0.81 (95% CI: 0.65-0.93) and specificity was 0.82 (95% CI: 0.61-0.94) for single APT imaging parameters. Conclusion: APT imaging showed high diagnostic performance in assessing treatment response in patients with post-treatment gliomas, and the addition of APT imaging to other advanced MRI techniques can improve the diagnostic accuracy for distinguishing TR from TE.

Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections.

Acute respiratory infections are widespread in vulnerable populations of all ages and are characterized by a variety of symptoms. The underlying infection can be caused by a multitude of microorganisms, including viruses and bacteria. Early detection of respiratory infections through rapid pathogen screening is vital in averting infectious respiratory disease epidemics. This study utilized a multiplex real-time PCR system to develop a three-tube reverse transcription-PCR (RT-PCR) assay, enabling simultaneously detect nine respiratory pathogens, including: influenza A and B, adenovirus, respiratory syncytial virus (RSV), Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Chlamydia pneumoniae, and Mycoplasma pneumoniae. This technique utilizes a one-step assay, with specifically designed TaqMan primer-probe sets combined in the same tube. This assay provided rapid and simplified detection of the nine prevalent pathogens, as well as increased sensitivity and reduced cross-contamination. This assay was evaluated using 25 related viral/bacterial strains as positive references, the other 25 irrelevant strains as negative controls, and clinical specimens from 179 patients. All positive strains were detected with no amplification of the non-target microorganism mixtures and the assay's detection limits ranged between 250-500 copies/ml (1.25-2.5 copies/reaction). A total of 167 (93.3%) samples tested positive for at least one of the pathogens identified; 109 of these samples were from patients confirmed to have RSV infections. The diagnostic accuracy of our assay was further confirmed by matching results from classical direct immunofluorescence assay and nucleotide sequencing. These data demonstrate the innovative multiplex real-time PCR assay as a promising alternative to the current approaches used for early screening of acute respiratory infections.

High Power and Large-Energy Pulse Generation in an Erbium-Doped Fiber Laser by a Ferromagnetic Insulator-Cr2Si2Te6 Saturable Absorber.

Large-energy mode-locked fiber lasers are extensively studied due to their indispensable use in various fields and applications. Recently, ferromagnetic insulators have attracted tremendous research interest in ultra-fast photonics because of their unique ferromagnetic properties and typical layered structure. In our work, Cr2Si2Te6 nanosheets are prepared and utilized as a saturable absorber (SA) in a large-energy mode-locked erbium-doped fiber (EDF) laser. With a total cavity length of 240 m, a stable mode-locked operation characterized by maximum pulse energy as high as 244.76 nJ with a repetition rate of 847.64 kHz is achieved. When the cavity length is extended to 390 m, the output maximum pulse energy is successfully scaled up to 325.50 nJ. To our knowledge, this is the largest pulse energy and highest output power level to be achieved in mode-locked fiber lasers by two-dimensional (2D) material saturable absorbers (SAs) so far. This work not only makes a forward step to the investigation of the generation of large-energy pulses in mode-locked fiber lasers but also fully proves that the ferromagnetic insulator-Cr2Si2Te6 possesses an excellent nonlinear absorption property, antioxidant capacity in ambient conditions, as well as outstanding thermal stability, which enriches our insight into 2D materials.

Current State and Progress of Research on the Role of lncRNA in HBV-Related Liver Cancer.

Hepatocellular carcinoma (HCC) is a malignant tumor with the highest mortality rate in the world, and hepatitis B virus (HBV) plays an important role in its development. Long noncoding RNA (lncRNA) is highly related to the inactivation of tumor suppressor genes and the activation of oncogenes in HCC. Researchers have used high-throughput sequencing technology to identify many noncoding transcripts related to the development of HCC and have studied the interaction between these transcripts and DNA, RNA, or protein to determine the relevant mechanism in the development of HCC. In general, the research on lncRNA represents a new field of cancer research, and the imbalance in lncRNA plays an pivotal role in the occurrence of liver cancer. In this review, we summarize some of the dysfunctional lncRNAs in human HCC associated with HBV infection. Their regulatory pathways, functions, and potential molecular mechanisms in the occurrence and development of HCC are discussed.

Artificial intelligence-powered microfluidics for nanomedicine and materials synthesis.

Artificial intelligence (AI) is an emerging technology with great potential, and its robust calculation and analysis capabilities are unmatched by traditional calculation tools. With the promotion of deep learning and open-source platforms, the threshold of AI has also become lower. Combining artificial intelligence with traditional fields to create new fields of high research and application value has become a trend. AI has been involved in many disciplines, such as medicine, materials, energy, and economics. The development of AI requires the support of many kinds of data, and microfluidic systems can often mine object data on a large scale to support AI. Due to the excellent synergy between the two technologies, excellent research results have emerged in many fields. In this review, we briefly review AI and microfluidics and introduce some applications of their combination, mainly in nanomedicine and material synthesis. Finally, we discuss the development trend of the combination of the two technologies.

microRNAs, the Link Between Dengue Virus and the Host Genome.

Dengue virus (DENV) is a small envelope virus of Flaviviridae that is mainly transmitted by Aedes aegypti and Aedes albopictus. It can cause dengue fever with mild clinical symptoms or even life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). At present, there are no specific drugs or mature vaccine products to treat DENV. microRNAs (miRNAs) are a class of important non-coding small molecular RNAs that regulate gene expression at the post-transcriptional level. It is involved in and regulates a series of important life processes, such as growth and development, cell differentiation, cell apoptosis, anti-virus, and anti-tumor. miRNAs also play important roles in interactions between host and viral genome transcriptomes. Host miRNAs can directly target the genome of the virus or regulate host factors to promote or inhibit virus replication. Understanding the expression and function of miRNAs during infection with DENV and the related signal molecules of the miRNA-mediated regulatory network will provide new insights for the development of miRNA-based therapies.

Identification of SARS-CoV-2 Variants and Their Clinical Significance in Hefei, China.

The ongoing coronavirus disease 2019 (COVID-19) pandemic represents one of the most exigent threats of our lifetime to global public health and economy. As part of the pandemic, from January 10 to March 10, 2020, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) began to spread in Hefei (Anhui Province, China) with a total of 174 confirmed cases of COVID-19. During this period, we were able to gather critical information on the transmission and evolution of pathogens through genomic surveillance. Particularly, the objective of our study was to track putative variants of SARS-CoV-2 circulating in Hefei for the first time and contribute to the global effort toward elucidating the molecular epidemic profile of the virus. Patients who showed symptoms of COVID-19 were routinely tested for SARS-CoV-2 infections via RT-PCR at the First Affiliated Hospital of Anhui Medical University. Whole-genome sequencing was performed on 97 clinical samples collected from 29 confirmed COVID-19 patients. As a result, we identified a local novel single-nucleotide polymorphism site (10,380) harboring a G → T mutation (Gly → Val) in Hefei. Further phylogenetic network analysis with all the sequences of SARS-CoV-2 deposited in GenBank collected in East and Southeast Asia revealed a local subtype of S-type SARS-CoV-2 (a1) harboring a C → T synonymous mutation (Leu) at position 18,060 of ORF1b, likely representing a local SARS-CoV-2 mutation site that is obviously concentrated in Hefei and the Yangtze River Delta region. Moreover, clinical investigation on the inflammatory cytokine profile of the patients suggested that mutations at positions 18,060 (the shared variable site of subtype a1) and 28,253(harboring a C → T synonymous mutation, Phe) were associated with milder immune responses in the patients.

Differential dynamics of peripheral immune responses to acute SARS-CoV-2 infection in older adults.

In this study, peripheral blood mononuclear cells from young and old patients with COVID-19 were examined phenotypically, transcriptionally and functionally to reveal age-, time- and severity-specific adaptations. Gene signatures within memory B cells and plasmablasts correlated with reduced frequency of antigen-specific B cells and neutralizing antibodies in older patients with severe COVID-19. Moreover, these patients exhibited exacerbated T cell lymphopenia, which correlated with lower plasma interleukin-2, and diminished antigen-specific T cell responses. Single-cell RNA sequencing revealed augmented signatures of activation, exhaustion, cytotoxicity and type I interferon signaling in memory T and natural killer cells with age. Although cytokine storm was evident in both age groups, older individuals exhibited elevated levels of myeloid cell recruiting factors. Furthermore, we observed redistribution of monocyte and dendritic cell subsets and emergence of a suppressive phenotype with severe disease, which was reversed only in young patients over time. This analysis provides new insights into the impact of aging on COVID-19.

Development and validation of a droplet digital PCR assay for the evaluation of PML-RARα fusion transcripts in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is an aggressive disease that requires prompt treatment. Promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion genes resulting from reciprocal translocation are considered a molecular basis for diagnosing APL. Moreover, PML-RARα fusion gene testing is an essential tool for monitoring the response to therapy via minimal residual disease and providing a diagnosis before rapid disease progression in APL. The present study developed a novel droplet digital PCR (ddPCR) assay to rapidly detect two PML-RARα variants (bcr1 and bcr3) and compared its limit of detection (LOD) with quantitative PCR (qPCR). It was demonstrated that the LOD of ddPCR for PML-RARα reached 0.001%, and the evaluation of high copy number samples of PML-RARα by ddPCR correlated well with qPCR. Furthermore, clinical sample testing with ddPCR found that 34 and 24% samples were bcr-1-positive and bcr3-positive, respectively. However, according to qPCR, 30% of the samples were bcr1-positive and 20% were bcr3-positive. In addition, the concordance rate between ddPCR and qPCR reaction was 86%. While monitoring minimal residual disease, the PML-RARα mutation rate of three patients who recovered well decreased to 0.34%. However, one patient who was bcr3-positive and relapsed had a mutation rate of 13% while in remission, indicating that the bcr3 isoform may be an adverse prognostic factor affecting recovery. Therefore, the present results suggested that this novel ddPCR assay may be useful for monitoring and evaluating the treatment effects and prognosis of APL.

Design and Evaluation of a Method for Testing Polymorphisms of Folate-Related Genes Using the Luminex Liquichip System.

Purpose: The methylene tetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C, and the methionine synthase reductase (MTRR) A66G polymorphisms are the three most common folate metabolism-related loci in the Chinese population. They are associated with numerous birth defects or congenital diseases. To facilitate screening and genetic counseling, we established a method for the simultaneous detection of these three polymorphisms using the Luminex liquid suspension chip and multiple asymmetric polymerase chain reactions (PCRs). Materials and Methods: The three polymorphisms were amplified by multiplex PCR with biotinylated primers, followed by hybridization with six probe-linked magnetic microspheres. The mean fluorescent intensity value in each microsphere was detected by Luminex Magpix for polymorphism detection in 150 samples and confirmed by sequencing. Results: The consistency between the Luminex liquid suspension chip method and sequencing was 100%. Among the 150 randomized samples, the minor allele frequency (MAF) of MTHFR C677T was 0.41, which was the most common variant allele, followed by MTRR A66G (MAF = 0.24), and finally MTHFR A1298C (MAF = 0.19). Conclusion: The Luminex liquid suspension chip method can replace sequencing to analyze the MTHFR C677T, MTRR A1298C, and MTRR A66G loci simultaneously as a rapid, convenient, accurate, and stable method for large-scale testing.

Evaluation of EGFR mutations in NSCLC with highly sensitive droplet digital PCR assays.

Targeted drugs have been widely used in the treatment of patients with lung cancer, particularly for those with non­small cell lung cancer (NSCLC). Plasma cell­free DNA is an emerging clinical tool for the detection of epidermal growth factor receptor (EGFR) gene mutation in patients with lung cancer. Detection of circulating tumor (ct) DNA by droplet digital PCR (ddPCR) is a highly sensitive and minimally invasive alternative for the assessment and management of cancer. In the present study, four ddPCR systems were developed to detect the 19DELs, L858R, T790M and C797S mutations of the EGFR gene in plasma ctDNA samples, and all exhibited higher sensitivity compared with the amplification refractory mutation system (ARMS)­PCR assays. The results revealed that the sensitivity of the ddPCR assays for the four major types of EGFR mutant reached 0.04%. In total, 50 plasma ctDNA samples were collected from patients with NSCLC to detect the 19DELs, L858R, T790M and C797S mutations by ddPCR and ARMS­PCR. All the mutations except for C797S were detected and the concordance rates between ddPCR and ARMS­PCR were 96% (19DELs), 98% (L858R) and 100% (T790M). The fraction of EGFR mutation ranged from 0.43 to 68.07% using the ddPCR method. Therefore, the present study suggests that the four ddPCR testing systems could be used for early detection of EGFR mutations in plasma samples, so that patients can better select the targeted drugs according to the EGFR mutation.