Detection Of Genomic DNA Research Articles

Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of Leishmania

To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.

JMM Profile: Bordetella pertussis and whooping cough (pertussis): still a significant cause of infant morbidity and mortality, but vaccine-preventable.

Whooping cough (pertussis) is a highly contagious respiratory bacterial infection caused by Bordetella pertussis and is an important cause of morbidity and mortality worldwide, particularly in infants. Bordetella parapertussis can cause a similar, but usually less severe pertussis-like disease. Bordetella pertussis has a number of virulence factors including adhesins and toxins which allow the organism to bind to ciliated epithelial cells in the upper respiratory tract and interfere with host clearance mechanisms. Typical symptoms of pertussis include paroxysmal cough with characteristic whoop and vomiting. Severe complications and deaths occur mostly in infants. Laboratory confirmation can be performed by isolation, detection of genomic DNA or specific antibodies. Childhood vaccination is safe, effective and remains the best control method available. Many countries have replaced whole-cell pertussis vaccines (wP) with acellular pertussis vaccines (aP). Waning protection following immunisation with aP is considered to be more rapid than that from wP. Deployed by resource-rich countries to date, maternal immunisation programmes have also demonstrated high efficacy in preventing hospitalisation and death in infants by passive immunisation through transplacental transfer of maternal antibodies.

The application of DNA-HRP functionalized AuNP probes in colorimetric detection of citrus-associated Alternaria genes

The monitoring of the fungal genus Alternaria, which causes destructive brown spot disease in citruses worldwide and produces highly toxic mycotoxins, is extremely important to protect citrus and human health. In this work, we describe an ultrasensitive colorimetric method for the detection of genomic DNA of Alternaria from citrus fruit samples, using a system consisting of five groups of reporter probes. Each reporter probe is prepared by coupling recognition DNA and horseradish peroxidase (HRP) on the surface of gold nanoparticle (AuNP) through a convenient and low-cost freezing-assisted method. Meanwhile, the capture DNA is immobilized on magnetic bead (MB) via biotin-streptavidin reaction. Then, the capture DNA, target DNA, and five groups of AuNP-based reporter probes form a stable DNA-heptamer sandwich structure on the MB, and then HRP generates a blue signal for the subsequent colorimetric detection. It should be noted that AuNP with a large specific surface area drives abundant HRP anchoring, resulting in significant signal amplification. In addition, there are five groups of AuNP-based reporter probes, which further amplify the detection signal. As a result, the detection limit of the artificial target DNA is as low as 15.6 pM. Because the detection signal can be recorded visually without any special equipment, and its sensitivity is high, this method represents a suitable diagnostic tool for Alternaria genetic detection.

An assessment of the epidemiology and socioeconomic impact of the 2019 African swine fever outbreak in Ngara district, western Tanzania

African swine fever (ASF) is a contagious viral transboundary animal disease affecting domestic pigs caused by ASF virus (ASFV). This study was conducted in order to determine the genetic characteristics, risk factors and socioeconomic impact of an ASF outbreak in 2019 in Ngara, western Tanzania. Tissue samples from dead domestic pigs with clinical picture suggestive of ASF infection were collected for ASF confirmation and genetic characterization of ASFV. Data on the risk factors and socioeconomic impact associated with the ASF outbreak were collected from consenting farmers using a semi-structured questionnaire. Disease confirmation was done by detection of genomic ASFV DNA using polymerase chain reaction. Partial amplification of the ASFV genome, dideoxynucleotide sequencing of the PCR products followed by bioinformatics analyses was conducted to determine the ASFV genotypes. Phylogenetic reconstruction of the variable 3′-end of the B646L gene clustered the ASFV isolate into genotype X. Analysis of the intergenic fragment sequences between 173R and 1329L genes showed that the viral strains TAN/19/Ngara and Kenya 1950 similarly lacked a 36 bp fragment that is present in strain Ken05/Tk1. Feeding pigs of uncooked swill was shown to be significantly associated with ASF spread (OR = 3.08, C.I.95% = 1.06–8.99, P = 0.0009). Occurrence of ASF outbreak resulted in loss of income and investment as most farmers kept pigs for the purpose of income generation. Food security was disturbed due to high pig mortality following occurrence of ASF outbreak. A total of 93,630,000 Tanzanian shillings (approximately 41,065 USD) was estimated to be lost as a result of pigs’ mortality in 219 households. The findings of the present study associate ASFV genotype X with the 2019 ASF outbreak in Ngara and feeding pigs with uncooked swill with spread of the disease.

Ninhydrin as a novel DNA hybridization indicator applied to a highly reusable electrochemical genosensor for Candida auris

This work reports a simple strategy for Candida auris genomic DNA (gDNA) detection, a multi-resistant fungus associated with nosocomial outbreaks in healthcare settings, presenting high mortality and morbidity rates. The platform was developed using gold electrode sensitized with specific DNA capture probe and ninhydrin as a novel DNA hybridization indicator. The genosensor was able to detect C. auris in urine sample by differential pulse voltammetry and electrochemical impedance spectroscopy. The biosensor's analytical performance was evaluated by differential pulse voltammetry, detecting up to 4.5 pg μL−1 of C. auris gDNA in urine (1:10, V/V). Moreover, the genosensor was reused eight times with no loss in the current signal response. The genosensor showed selectivity and stability, maintaining 100% of its response up to 80 days of storage. In order to analyze interactions of single and double-stranded DNA with ninhydrin, SEM, AFM and molecular dynamics studies followed by docking simulations were performed. Theoretical calculations showed ninhydrin interactions more favorably with dsDNA in an A-T rich binding pocket rather than with the ssDNA. Therefore, the proposed system is a promising electrochemical detection device towards a more accurate detection of C. auris gDNA in biological samples.

Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH.

The sequence-specific recognition of duplex DNA by unmodified parallel triplex-forming oligonucleotides is restricted to low pH conditions due to a necessity for cytosine protonation in the third strand. This has severely restricted their use as gene-targeting agents, as well as for the detection and/or functionalisation of synthetic or genomic DNA. Here I report that the nucleobase 6-amino-5-nitropyridin-2-one (Z) finally overcomes this constraint by acting as an uncharged mimic of protonated cytosine. Synthetic TFOs containing the nucleobase enabled stable and selective triplex formation at oligopurine-oligopyrimidine sequences containing multiple isolated or contiguous GC base pairs at neutral pH and above. Moreover, I demonstrate a universal strategy for the enzymatic assembly of Z-containing TFOs using its commercially available deoxyribonucleotide triphosphate. These findings seek to improve not only the recognition properties of TFOs but also the cost and/or expertise associated with their chemical syntheses.

Rapid and Sensitive Point of Care Detection of MRSA Genomic DNA by Nanoelectrokinetic Sensors

Biosensors have shown great potential in realizing rapid, low cost, and portable on-site detection for diseases. This work reports the development of a new bioelectronic sensor called AC electrokinetics-based capacitive (ABC) biosensor, for the detection of genomic DNA (gDNA) of methicillin-resistant Staphylococcus aureus (MRSA). The ABC sensor is based on interdigitated microelectrodes biofunctionalized with oligonucleotide probes. It uses a special AC signal for direct capacitive monitoring of topological change on nanostructured sensor surface, which simultaneously induces dielectrophoretic enrichment of target gDNAs. As a result, rapid and specific detection of gDNA/probe hybridization can be realized with high sensitivity. It requires no signal amplification such as labeling, hybridization chain reaction, or nucleic acid sequence-based amplification. This method involves only simple sample preparation. After optimization of nanostructured sensor surface and signal processing, the ABC sensor demonstrated fast turnaround of results (~10 s detection), excellent sensitivity (a detection limit of 4.7 DNA copies/µL MRSA gDNA), and high specificity, suitable for point of care diagnosis. As a bioelectronic sensor, the developed ABC sensors can be easily adapted for detections of other infectious agents.

Combination of ferrocene decorated gold nanoparticles and engineered primers for the direct reagentless determination of isothermally amplified DNA.

A reagent-less DNA sensor has been developedexploiting a combination of gold nanoparticles, modified primers, and isothermal amplification. It is applied to the determination ofKarlodinium armiger, a toxic microalgae, as a model analyte to demonstrate this generic platform. Colloidal gold nanoparticles with an average diameter of 14 ± 0.87nm were modified with a mixed self-assembled monolayer of thiolated 33-mer DNA probes and (6-mercaptohexyl) ferrocene. Modified primers, exploiting a C3 spacer between the primer-binding site and an engineered single-stranded tail, were used in an isothermal recombinase polymerase amplification reaction to produce an amplicon by two single-stranded tails. These tails were designed to be complementary to a gold electrode tethered capture oligo probe, and an oligo probe immobilized on the gold nanoparticles, respectively. The time required for hybridization of the target tailed DNA with the surface immobilized probe and reporter probe immobilized on AuNPs was optimized and reduced to 10min, in both cases. Amplification time was further optimized to be 40min to ensure the maximum signal. Under optimal conditions, the limit of detection was found to be 1.6 fM of target dsDNA. Finally, the developed biosensor was successfully applied to the detection of genomic DNA extracted from a seawater sample that had been spiked with K. armiger cells. The demonstrated generic electrochemical genosensor can be exploited for the detection of any DNA sequence and ongoing work is moving towards an integrated system for use at the point-of-need.

A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics.

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas-powered catalytic nucleic acid circuit, namely, CRISPR-Cas-only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

Establishment of a duplex real-time qPCR method for detection of Salmonella spp. and Serratia fonticola in fishmeal

Salmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratia fonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/μL and 145 copies/μL, respectively (correlation coefficient R2 = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of fishmeal was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.

Serial Plasma Cell-Free Circulating Tumor DNA Tests Identify Genomic Alterations for Early Prediction of Osimertinib Treatment Outcome in EGFR T790M–Positive NSCLC

IntroductionRecent advances in the detection of genomic DNA from plasma samples allow us to follow tumor DNA shedding in plasma during systemic treatment. Osimertinib is the standard of care for patients with NSCLC with acquired EGFR T790M mutations. We assessed changes in serial plasma cell-free circulating tumor DNA (ctDNA) genomic alterations to predict osimertinib efficacy. MethodsWe prospectively collected plasma from patients having EGFR-mutated advanced NSCLC previously treated with EGFR tyrosine kinase inhibitor therapy and with acquired EGFR T790M mutation detected by standard methods. Plasma samples were collected before starting osimertinib treatment, 4 weeks after osimertinib treatment, and on progression. ctDNA was analyzed using the Guardant360 assay. ResultsA total of 15 eligible patients received osimertinib. Before starting treatment, EGFR-activating mutations were detected in the ctDNA of all patients, and EGFR T790M was detected in 93% of the cases. Osimertinib treatment was associated with an objective response rate of 53% and a median progression-free survival of 7.3 months. A total of 12 of the 15 patients had undetectable plasma T790M and decreased activating mutation allelic frequency (AF) at week 4. None of the 12 patients had disease progression within 16 weeks. For the remaining three patients, with detectable plasma T790M (n = 2) or increased activating mutation AF (n = 1) at week 4, two had progressive disease within 16 weeks (p = 0.03). ConclusionsIn patients with EGFR-mutated advanced NSCLC, persistent EGFR T790M or increasing activating mutation AF as detected in ctDNA 4 weeks after the start of osimertinib treatment may predict disease progression within 16 weeks.

An impedimetric genosensor for Leishmania infantum based on electrodeposited cadmium sulfide nanosheets

It is estimated that there are 400000 new cases of visceral leishmaniasis each year, with about 30,000 deaths. Therefore, detection of this pathogen and its species is highly vital for overall health of the community. In the present research, a DNA-based biosensor, namely genosensor, was introduced for detection of genomic DNA of Leishmania infantum. The genosensor was fabricated based on the transduction of cadmium sulfide nanosheets and recognition of a particular single stranded DNA sequence, and worked in label-, marker-, tag- and PCR-free manners. Impedimetric measurements were performed in a wide range of frequency (recording Nyquist diagrams) without applying external force (working at open circuit potential) upon hybridization of DNA targets with the cadmium sulfide nanosheets surface-immobilized probe. The genosensor detected the complementary DNA strand in a concentration range of 1.0 × 10−14 to 1.0 × 10−6 mol L−1 and a detection limit (DL) of 0.81 fmol L−1 (6.5 fg mL−1), and genomic DNA of Leishmania infantum in a concentration range of 5–50 ng μL−1 and a DL of 1.2 ng μL−1. The genosensor had a very good selectivity, fabrication reproducibility and stability, and was applicable for practical applications.

Fluorometric Detection of Low-Abundance EGFR Exon 19 Deletion Mutation Using Tandem Gene Amplification.

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application.

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

Quantum scale organic semiconductors for SERS detection of DNA methylation and gene expression

Cancer stem cells (CSC) can be identified by modifications in their genomic DNA. Here, we report a concept of precisely shrinking an organic semiconductor surface-enhanced Raman scattering (SERS) probe to quantum size, for investigating the epigenetic profile of CSC. The probe is used for tag-free genomic DNA detection, an approach towards the advancement of single-molecule DNA detection. The sensor detected structural, molecular and gene expression aberrations of genomic DNA in femtomolar concentration simultaneously in a single test. In addition to pointing out the divergences in genomic DNA of cancerous and non-cancerous cells, the quantum scale organic semiconductor was able to trace the expression of two genes which are frequently used as CSC markers. The quantum scale organic semiconductor holds the potential to be a new tool for label-free, ultra-sensitive multiplexed genomic analysis.

Design and fabrication of PDMS microfluidics device for rapid and label-free DNA detection

Microfluidics explores the manipulation of fluid in small volume, a multidisciplinary field is imperative for DNA extraction. This study offers a simple yet substantial methodology for the fabrication of microfluidics structure-based polydimethylsiloxane (PDMS) biopolymer on a glass substrate with SU-8 photoresist for label-free detection of pathogenic genomic DNA. Two microfluidics designs for DNA detection were based on AutoCAD software, both contain two inlets and one outlet, with dimensions of 28 mm wide, and 18 mm long, and total surface area of 504 mm2. The designs were patterned in such particular sizes and dimensions to test fluid delivery and enhancement in biochemical processes in DNA extraction, while maintaining economical values as a disposable chip. Both microfluidics devices showed no leakage during fluid delivery, have heights of 97.97 and 103.44 μm, and surface roughness of 0.15 and 0.07 μm, respectively. DNA extraction from pathogenic fungus Ganoderma boninense was run on PDMS microfluidic device and UV–Vis analysis confirmed successful extraction with peaks found at 260–280 nm. Current–voltage (I–V) measurement confirmed the accuracy of microfluidic device for the specific pathogen with both real and synthetic samples of G. boninense illustrating the similar graph values of only 0.000005 A difference at 1.0 V after hybridization.

Development of Leuconostoc lactis–Specific Quantitative PCR and its Application for Identification and Enumeration in Fermented Foods

Leuconostoc lactis plays a pivotal role in providing a pleasant aroma in fermented foods. In this study, an L. lactis-specific quantitative PCR (qPCR) method was developed by designing species-specific primers using comparative pan-genomic analysis to monitor the microbial populations during food fermentation. As a result, a hypothetical protein-encoding gene, Llac_03470, was selected as a target gene to detect L. lactis using a designed set of primers (NlacF and NlacR). PCR analysis against genomic DNA of various lactic acid bacteria confirmed the specificity of the primers for the detection of genomic DNA of L. lactis strains. By using the primer set, a standard curve for qPCR analysis of L. lactis was established, and no significant differences (p < 0.05) were found in the yields of the genomic DNA recovered from MRS medium and kimchi. In addition, the qPCR was validated to successfully enumerate L. lactis during kimchi fermentation. In conclusion, the newly developed qPCR method in this study can be used to detect L. lactis in fermented kimchi and to monitor the change in its population during the fermentation process.

Method for RNA extraction and transcriptomic analysis of single fungal spores

Transcriptomic analysis of single cells has been increasingly in demand in recent years, thanks to technological and methodological advances as well as growing recognition of the importance of individuals in biological systems. However, the majority of these studies have been performed in mammalian cells, due to their ease of lysis and high RNA content. No single cell transcriptomic analysis has yet been described in microbial spores, even though it is known that heterogeneity at the phenotype level exists among individual spores. Transcriptomic analysis of single spores is challenging, in part due to the physically robust nature of the spore wall. This precludes the use of methods commonly used for mammalian cells. Here, we describe a simple method for extraction and amplification of transcripts from single fungal conidia (asexual spores), and its application in single-cell transcriptomics studies. The method can also be used for studies of small numbers of fungal conidia, which may be necessary in the case of limited sample availability, low-abundance transcripts or interest in small subpopulations of conidia.•The method allows detection of transcripts from single conidia of Aspergillus niger•The method allows detection of genomic DNA from single conidia of Aspergillus niger

Genomic DNA detection frequently-used chemotherapeutic drugs and their adverse drug reactions in children with rhabdomyosarcoma

Objective To analyze and summarize the results of genomic DNA test findings of chemotherapeutic drugs commonly used in pediatric rhabdomyosarcoma (RMS) in children, and to analyze the relationship between adverse reactions to chemotherapy toxicity and genomic DNA polymorphisms, so as to provide evidence for guiding treatment. Methods Retrospective analysis was conducted in RMS children admitted at Hematology Oncology Center, Beijing Children′s Hospital, Capital Medical University from January 2017 to June 2018.The criteria for enrollment were definite diagnosis of RMS, regular treatment and follow-up at Hematology Oncology Center, Beijing Children′s Hospital, Capital Medical University, and detection of peripheral blood DNA fluorescence hybridization sequence for several commonly chemotherapy drugs.The toxicity of chemotherapeutic drugs was detected based on the National Cancer Institute routine toxicity criteria (NCI-CTCAE version 4.0). Summary and analysis indicators included primary and metastatic site, size, international RMS clinical stage (TNM-UICC), Intergroup Rhabdomyosarcoma Study(IRS) Clinical Grouping Classification, risk grouping, pathological type, changes in major organ functions, as well as processes of surgery, chemotherapy and radiotherapy, and the association between toxicity and DNA polymorphism of drug genes was analyzed.SPSS 22.0 software was used for χ2 test. Results A total of 32 children were enrolled, and 20 cases were male and 12 cases were female, their median age was 50 months (15-120 months). The primary tumor of 9 cases were sited in the chest, abdomen and basin, 8 cases in the head and neck (non-meningeal), 7 cases in bladder prostate, 3 cases in limbs, 2 cases in the meningeal area, 1 case in urogenital tract (non-bladder prostate), 2 cases in other parts.Seventeen cases were embryonic type and 15 cases were alveolar type.Five cases were TNM-Ⅰ stage, 5 cases were TNM-Ⅱ stage, 10 cases TNM -Ⅲ stage, 12 cases were TNM-Ⅳ stage, 21 cases were IRS-Ⅲ, 11 cases were IRS-Ⅳ.Twenty-two cases were moderate-risk (MR), 10 cases were high-risk (HR). Twenty-two cases were detected UGT1A1*6 gene, 18 cases in GG type, 13 cases in GA type, and 1 case in AA type.ABCB1 gene monitoring was performed in 27 children, 14 cases of CT type and 13 cases of TT type; 29 cases were detected GSTP1 gene, 7 cases of GA type and 2 cases of GG type, 19 cases of AA type, 1 case of AG type; 30 cases were detected CYP3A5 gene, 2 cases of GA type, 13 cases of GG type, AG 15 cases.All patients were treated according to the BCH-RMS-2007 protocol using VAC (Vincristine, Doxorubicin, and Cyclophosphamide) as the basis for chemotherapy.From 2017, VAC and VI regimen (Vincristine, Irinotecan) were defined as the standard of backbone chemotherapeutic regimen for MR.Nine cases underwent surgery before chemotherapy and 10 cases had surgery after chemotherapy, among them, 5 cases underwent twice operation.Local radiotherapy was performed on the 12th week of chemotherapy, and the central nervous system involvement cases started in the first week.Hematological toxicity was mainly caused by neutropenia, with 2 cases of grade 3 and 30 cases of grade 4.Liver function damage of grade 2 was 6 cases, grade 3 was 3 cases.Four patients with grade 1 diarrhea, 3 patients with grade 2, 5 patients with grade 3, 3 patients with grade 4.There was significant diffe-rence between the severity of diarrhea and UGT1A1*6 genotype polymorphism(P<0.05). Conclusions Chemothe-rapy for RMS patients is highly safety.If the genomic DNA test of chemotherapy drugs show a slow metabolism type, the dose of chemotherapy should be reduced, and the toxicity of chemotherapy drugs should be monitored dynamically. Key words: Rhabdomyosarcoma; Side effect; Pharmacogenomics; Irinotecan

E-DNA biosensors of M. tuberculosis based on nanostructured polypyrrole.

The objective of this paper is to demonstrate the potential of nanostructured polypyrrole formed by template free as platform for amperometric detection of DNA. The nanowires of polypyrrole (nw-PPy) are formed through electrochemical polymerization and chemically modified by electrochemical oxidation of ethylene diamine or dendrimers PAMAM to obtain aminated surface. The DNA probe and ferrocenyl group, as redox reporter, were covalently linked to the surface of nw-PPy. The chemical structure of nanostructured platform was characterized through SEM, FT-IR and XPS and the electrochemical properties through cyclic voltammetry and electrochemical impedance spectroscopy (EIS). We show that the properties of nw-PPy such as, hydrophilic character and large surface area have large effect on the electronic properties. Thus, the electrochemical performance is increased compared to others nanomaterials considering the obtained value of the rate of electron transfer of 18 s-1. These properties allow enhanced DNA sensing where detection limit of 0.36 atomolar without any amplification step. The biosensor can be applied in detection of genomic DNA of Mycobacterium tuberculosis and the mutated one which present the resistance to rifampicin and large selectivity was demonstrated. We believe that nw-PPy modified with redox marker is a promising platform for electrochemical biosensors and can be applied for various diagnosis prospects.