Probe Amplification Research Articles

A real-time immunocapture PCR (RT-IPCR) without interference of protein A for convenient detection of staphylococcal enterotoxin B from food and environmental samples

PurposeA real-time immunocapture PCR (RT-IPCR) has been fabricated for the detection of Staphylococcus aureus enterotoxin B (SEB) from food and environmental samples.MethodsConsidering the fact, anti-SEB immunoglobulin G (IgG) has affinity towards protein A, produced by nearly all S. aureus, and generates false-positive read out in all immuno-based assay. We have employed avian anti-SEB antibody (SEB-IgY) as capture probe, since IgY interact less efficiently to protein A and biotinylated SEB-specific monoclonal antibody (SEB -MAb) conjugated with reporter DNA as revealing probe for real-time PCR amplification and signal generation. Sensitivity and selectivity of the assay were evaluated employing closely related enterotoxins and other toxins.ResultsThe RT-IPCR is highly specific and sensitive (100 fg/mL). The practical applicability of the assay was tested using spiked food sample as well as naturally contaminated food samples. The sensitivity and specificity of RT-IPCR were not compromised by the foods tested and was able to detect SEB conveniently. Further, the assay was validated comparing with the in-house developed PCR, and plausible result was obtained.ConclusionThe developed assay can be utilized as a low-cost detection system of SEB in routine food testing laboratories.

Electrochemical detection of Mycobacterium tuberculosis IS6110 gene fragments based on the gold nanocrystals with uniform morphology and highly exposed high-index facets and target DNA-induced recycling amplification

This paper reports the synthesis of gold nanocrystals via the reduction of chloroauric acid using ascorbic acid in the mixed hexadecyltrimethylammonium chloride aqueous solution with potassium bromide and potassium iodide. The resulting gold nanocrystals have trioctahedral nanostructures with uniform morphology and highly exposed high-index facets. The nanocrystals unique structure promotes enhanced chemical activity and catalytic activity. Preparation of the electrochemical sensing platform for IS6110 gene involves the modification of gold nanocrystals on hairpin DNA 2 (H2) and thionine as the redox probe for target DNA-induced signal amplification. Target DNA (IS6110 gene) hybridizes with hairpin DNA 1 (H1) pre-modified on the gold electrode, which opens the hairpin structure of H1. This can hybridize with H2 to release target DNA, which is used in the next recycle. The utilization of target DNA-induced recycling allows one target DNA to transport many redox probes to the electrode surface promoting significant signal amplification. The detection signal is further enhanced by catalyzed redox reaction of thionine with gold nanocrystals. The differential pulse voltammetric signal increases with increasing concentration of target DNA in the range of 0.1-1.0 × 105 fM with detection limit of 0.031 fM. The method provides ultrahigh sensitivity, specificity and stability and was successfully applied in the detection of tuberculosis in human blood.

How to Perform miRacles: A Step-by-Step microRNA Detection Protocol Using DNA Nanoswitches.

MicroRNAs are short non-coding RNAs involved in post-transcriptional gene regulation, and are increasingly considered to be biomarkers for numerous biological processes and human diseases. Current techniques used for microRNA detection can be expensive and labor-intensive, and typically require amplification, labeling, or radioactive probes. In this protocol, we describe a DNA nanoswitch-based microRNA detection assay termed "miRacles": microRNA-activated conditional looping of engineered switches. This method uses conformationally responsive DNA nanoswitches that detect the presence of specific microRNAs with a simple and unambiguous gel-shift assay that can be performed on the benchtop. The assay is low cost, minimalistic, and capable of direct detection of specific microRNAs in unprocessed total RNA samples, with no enzymatic amplification, labeling, or special equipment. The protocol for detection of microRNAs in total RNA can be completed in as little as a few hours, making this assay a compelling alternative to qPCR and Northern blotting. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of DNA nanoswitches Basic Protocol 2: Detection of microRNAs from total RNA samples Support Protocol 1: Optional nanoswitch purification by PEG precipitation Support Protocol 2: Optional nanoswitch purification by liquid chromatography.

Human papillomavirus infection maybe not associated with primary lung cancer in the Fujian population of China.

BackgroundTo investigate whether human papillomavirus (HPV) infection is associated with primary lung cancer among the Fujian population.MethodsHPV infection was detected in 140 pairs of lung cancer tissues and matched paracancerous tissues by examining the 21 clinically relevant HPV types using a combination of viral highly conserved L1 region PCR amplification and specific probe reverse hybridization. Paired χ2 test was used to analyze differences in detection rates of HPV between lung cancer and paracancerous tissues. Differences in detection rates of HPV in lung cancer tissues were analyzed using χ2 test or the exact probability method. The rank sum test was used to analyze differences in the distributions of routine indices of blood and pulmonary function in lung cancer tissues between the HPV negative and positive groups.ResultsHPV infection was detected in 13 of the 140 tumor specimens and in 16 of the paired normal lung tissues. There was no significant correlation between HPV infection and lung cancer (P > 0.05). The diagnosed HPV infection rates did not differ significantly among lung cancer tissues with different stratification (P > 0.05). However, the platelet count, platelet pressure, residual gas volume, functional residual volume, and residual gas volume/lung total distribution may differ between HPV‐negative and HPV‐positive lung cancer tissues (0.000625 < P < 0.05).ConclusionsWe concluded that HPV infection may not be associated with the risk of primary lung cancer in the Fujian population. However, HPV infection may affect platelet and residual lung function in primary lung cancer patients.

Analysis of rare thalassemia caused by HS-40 regulatory site deletion

ABSTRACT Objectives: To investigate the effect of HS-40 regulatory site deletion on α-globin gene expression and its clinical significance. Methods: Venous blood samples of subjects were analyzed using a hematology analyzer and high- performance liquid chromatography; fetal cord blood was analyzed by a capillary electrophoresis analyzer. Gap-polymerase chain reaction (PCR), reverse dot blot (RDB), and multiple-link-dependent probe amplification (MLPA) were used for genotyping of thalassemia. Results: The proband was POLR3 K, HS-40 heterozygous deletion; the proband’s wife was –SEA/αα; the fetus was POLR3 K, HS-40 heterozygous deletion combined with –SEA deletion; all of them had microcytic hypochromic anemia. Fetal umbilical cord blood electrophoresis revealed a suspected Hb Bart’s band to be 88.4%, and the fetus was, thus, diagnosed as Hb Bart’s fetus. The red blood cell parameters of the sporadic case showed that he had microcytic hypochromic anemia. Hemoglobin (Hb) electrophoresis analysis showed Hb H to be 5.3%, leading to a diagnosis of Hb H disease. Gap-PCR and RDB identified the genotype to be –α3.7/αα, βA/βA. MLPA detected heterozygous deletion or –α3.7 deletion on one allele and deletion of the HS-40 regulatory site on the other allele. Conclusion: The deletion of HS-40 regulatory site reduced expression of α-globin. HS-40 heterozygous deletion manifested as mild anemia, which was of microcytic hypochromic type. When compounded with –α3.7/αα, it manifested as Hb H disease; and when compounded with –SEA/αɑ, it manifested as Hb Bart’s fetus.

Deletion and duplication mutations spectrum in Duchenne muscular dystrophy in the southwest of Iran

BackgroundDuchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by the mutation within the dystrophin gene. Objectivesthe primary objective of this study was to evaluate the deletion and duplication mutations in DMD patients. Materials and methodsOur study has identified 33 Iranian patients collected from Narges Genetic lab, southwest of Iran, Ahvaz. All cases were subjected to complete clinical evaluation pedigree analysis, electromyography studies, estimation of serum creatine phosphokinase (CPK) level, and DNA analysis. DNA samples were analyzed by multiplex-ligation-dependent probe amplification (MLPA). ResultsIn this study, deletion rate was 75% (25/33) and more frequent in the distal end exons whereas duplication rate was 25% (8/33) and it is more frequent at proximal exons. Most of the deletion (15/25, 60%) were located on the distal hot spot region (exon 44–55). Majority of exon duplications 62.5% (5/8) were located at the proximal hot spot region (exon 1–16) and 37.5% (3/8) were located at the distal hot spot region (exon 50–62). Single exon deletions were present in 10/25 patients (40%) which are mostly were in exon 45, 51 and 52 with similar frequencies (2/25, 8% each). One patient had a de novo single exon deletion (exon 51) and whole dystrophin gene was deleted in only one patient. ConclusionThis finding indicates that as with deletions, duplications occur non-randomly but with a dramatically different distribution. Duplication frequency is highest near the 5′ end of the gene.

Primary hepatocellular adenoma due to biallelic HNF1A mutations and its co-occurrence with MODY 3: case-report and review of the literature

PurposeMaturity-onset diabetes of the young type 3 (MODY 3) is a consequence of heterozygous germline mutations in HNF1A, and a subtype of hepatocellular adenoma (HCA) is caused by biallelic somatic HNF1A mutations; rare HCA may be related to MODY 3. This study aimed to investigate the cosegregation of HNF1A mutations with diabetes and HCA in two families.MethodsTwo patients suffering from HCA and diabetes were screened for HNF1A germline and somatic mutations using direct sequence analysis and methylation-specific multiplex-ligation-dependent probe amplification (MS-MLPA) assay. Further, we screened eight relatives in the two independent families for diabetes, HCA and HNF1A variants. Additionally, we reviewed the literature concerning the phenotypes of MODY 3 and HCA at the background of HNF1A mutations.ResultsHere we reported two families (a total of six relatives) with two missense germline mutations of HNF1A identified initially using direct sequence analysis (c.686G>A in family A and c.526 + 1G>A in family B). Somatic deletion of the second allele of HNF1A was found in liver tumor tissues in both probands who were diagnosed with HCA. There are a total of ten cases of both MODY 3 and HCA phenotypes reported in the literature to date; incomplete penetrance for HCA was observed, and all the patients with HCA developed diabetes. The onset of diabetes and HCA was highly variable, the treatment of diabetes varied from diet to insulin, and the clinical expression of HCA ranged from silent to hemorrhage. Further, the severity of diabetes mellitus was not related to the occurrence of HCA.ConclusionsThis study describes the association of HCA and MODY 3 at the background of HNF1A mutations and highlights the importance of screening for HCA in MODY 3 families to avoid the possibility of severe complications. Further, the current study indicated that there may be a special mutational spectrum of HNF1A correlated with HCA in MODY 3 families.

A novel gross deletion and breakpoint junction sequence analysis of ATP7B in a Chinese family with Wilson disease using next‑generation sequencing and Sanger sequencing.

Wilson disease (WD) is a rare autosomal recessive genetic disorder that causes abnormal copper metabolism, resulting in pathological accumulation of copper in the liver, brain and other organs. Mutations in the ATPase copper transporter 7B (ATP7B) gene, which encodes a membrane P-type adenosine triphosphatase, have been identified as being responsible for WD. The present study analyzed clinical data and collected DNA samples from a pediatric patient with WD and her healthy parents. Mutation screening for ATP7B was performed using direct sequencing, multiplex ligation-dependent probe amplification(MLPA), next-generation sequencing (NGS) and Sanger sequencing of the breakpoint junction sequence. The patient (age, 2.7 years) presented with early-onset hepatic disease. The present study identified compound heterozygous mutations of ATP7B, including a heterozygous mutation (p.Arg1,041Trp) and a novel heterozygous gross deletion of a 57,771 bp fragment (chr13: 52490972-52548742) (GRCh37) from partial exon2- exon21 to external ATP7B sequence (15.833bp) in the patient. Analysis of the family members of the patient showed that the missense mutation and the gross deletion mutation were inherited from her mother and father, respectively. Microhomology and inverted repeat sequences, which may mediate the deletion mutation, were identified through sequence analysis on both sides of the breakpoints of this deletion. The present study provided additional information on the genotypic spectrum of the ATP7B gene, particularly with regard to early onset hepatic disease, as observed in the present patient with WD. The identification of the precise breakpoint junction sequence warrants further investigation of DNA break and recombination mechanisms. In detecting precise deletions, the NGS associated with Sanger sequencing of breakpoint junction sequence have been found to have more advantages than MLPA.

A ternary probe for target-triggered autonomous multi-branch rolling circle amplification for highly sensitive colorimetric sensing of platelet-derived growth factor BB

Highly sensitive detection of protein biomarkers has an important role in clinical diagnosis and disease treatment. On the basis of a newly designed ternary probe containing the aptamer, primer and circular DNA sequences, we have established an ultrasensitive and label-free colorimetric approach for detecting platelet-derived growth factor BB protein biomarkers via a target-triggered autonomous multi-branch rolling circle amplification (mbRCA) strategy. The target molecules specifically bind the aptamer sequences in the ternary probes and causes the configuration transition of the primers to trigger mbRCA generation of numerous of G-quadruplex sequences with the presence of the assistance hairpin. Hemin further associates with these G-quadruplexes to yield many hemin/G-quadruplex DNAzymes with the horseradish peroxidase-mimicking function, which catalyze the substrate solution to induce intense color transition for ultrasensitive and label-free colorimetric determination of the target molecules with a 0.055 pg mL−1 of detection limit in the range of 0.1–1000 pg mL−1. This aptamer-based sensing method exhibits high selectivity and can achieve the detection of low levels of protein biomarkers in diluted serum samples, which offers the developed approach new opportunities for detecting other trace protein biomarkers for the purpose of early disease diagnosis.

Highly sensitive and simultaneous detection of microRNAs in serum using stir-bar assisted magnetic DNA nanospheres-encoded probes

There are critical interests in the detection of microRNA (miRNA) because it can be a blood-borne biomarker, but analytical strategies are still limited by its small size, high sequence homology among family members and low abundance. In this work, three-dimensional magnetic DNA nanospheres were synthesized and immobilized on a gold stir-bar as encoded probes for miRNA capture and signal amplification. Electrochemical tags-labeled DNAs were immobilized on gold coated magnetic nanospheres via a hyperbranched hybridization chain reaction (HHCR). Subsequently, the magnetic DNA nanospheres were immobilized on the gold stir-bar as encoded probes. Target miRNAs were captured on the surface of the stir-bar by replacing the magnetic DNA nanospheres-encoded probes, and the probes were magnetically enriched for highly sensitive and selective electrochemical detection. The gold stir-bar assisted magnetic DNA nanospheres-encoded probes possess dual functions: They are as a nanocarrier to increase the loading amounts of HHCR products, and they are also a platform for efficient electrochemical signal amplification via magnetic enrichment. The method was successfully applied for the detection of miRNA21 and miRNA155 in a wide linear range of 5 fM to 2 nM, and with detection limits of 1.5 fM and 1.8 fM, respectively. The preliminary application of the method suggests that it has great potential in the detection of miRNAs in serum samples.

RETRACTED: 2D magnetic MoS2–Fe3O4 hybrid nanostructures for ultrasensitive exosome detection in GMR sensor

Exosome released from cells plays an important role in intercellular communication and show great clinical potential in early cancer screening and prognosis. Herein, an ultrasensitive Giant Magnetoresistance (GMR) biosensor was developed for exosome detection, which was based on 2D MoS2-Fe3O4 nanostructures (MOFE) as magnetic responsive probes for signal amplification. The MOFE can be readily synthesized with simple phase transfer method. Compared to pure Fe3O4 magnetic nanoparticles (MNPs), the layered MoS2 function as a template for recruiting high loading density of MNPs as magnetic probes. After modified by aptamer, we discover that the 2D magnetic MOFE hybrid nanostructures enable both multidentate targeting and multi-magnetic particle-based signal amplification, increasing the magnetic sensor performance, especially in sensitivity and output signal. Moreover, the 2D magnetic nanocomposites afford high selectivity and excellent reproducibility with detection limit of 100 exosomes in GMR sensor. Results demonstrate that the magnetic strategy based on 2D structures introduced here provide a new dimension for exosome detection, which show great potential of engineering 2D magnetic nanobioprobes for GMR based liquid biopsy application.

Electrochemical DNA sensor for inorganic mercury(II) ion at attomolar level in dairy product using Cu(II)-anchored metal-organic framework as mimetic catalyst

A sensitive and practical electrochemical DNA sensor is developed for evaluating attomolar mercury ion (Hg2+) in dairy product. Herein, the porous graphene oxide/gold nanoparticles (GO@Au) hybrids are exploited to construct high-conductive sensing interface. And importantly, Cu(II)-anchored metal-organic frameworks (MOFs) are prepared to design favorable signal probes for enzyme-free catalytic amplification. The results demonstrate that Cu(II)-anchored MOFs show excellent catalytic activity to glucose oxidation and offer an alternative mimetic catalyst to amplify the electrochemical signal. This approach attains an ultrasensitive assay of Hg2+ ranging from 0.10 aM to 100 nM with detection limit of 0.001 aM. Moreover, benefiting from thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry, the strategy exhibits good anti-jamming capability to distinguish Hg2+ from complex media, and thus could be applied for direct and rapid monitoring of Hg2+ in dairy product. The proposed sensor provides a valuable protocol with simple preparation, easy regeneration, high sensitivity and selectivity, indicating a promising prospect for on-site assessing quality deterioration in dairy product polluted with Hg2+.

EP10.20: Contribution of multiplex ligation‐dependent probe amplification (MLPA) panels in fetuses with congenital heart disease in Mexico: a pilot study

EP10.20: Contribution of multiplex ligation‐dependent probe amplification (MLPA) panels in fetuses with congenital heart disease in Mexico: a pilot study

New insights into genetic variant spectrum and genotype-phenotype correlations of Rubinstein-Taybi syndrome in 39 CREBBP-positive patients.

BackgroundRubinstein‐Taybi syndrome (RSTS) is a rare congenital disorder characterized by broad thumbs and halluces, intellectual disability, distinctive facial features, and growth retardation. Clinical manifestations of RSTS are varied and overlap with other syndromes’ phenotype, which makes clinical diagnosis challenging. CREBBP is the major causative gene (55%–60% of the cases), whereas pathogenic variants found in EP300 represent the molecular cause in 8% of RSTS patients. A wide range of CREBBP pathogenic variants have been reported so far, including point mutations (30%–50%) and large deletions (10%).MethodsThe aim of this study was to characterize the CREBBP genetic variant spectrum in 39 RSTS patients using Multiplex Ligation‐dependent Probe Amplification and DNA sequencing techniques (Sanger and Trio‐based whole‐exome sequencing).ResultsWe identified 15 intragenic deletions/duplications, ranging from one exon to the entire gene. As a whole, 25 de novo point variants were detected: 4 missense, 12 nonsense, 5 frameshift, and 4 splicing pathogenic variants. Three of them were classified as of uncertain significance and one of the patients carried two different variants.ConclusionSeventeen of the 40 genetic variants detected were reported for the first time in this work contributing, thus, to expand the molecular knowledge of this complex disorder.

Electrochemical determination of Salmonella typhimurium by using aptamer-loaded gold nanoparticles and a composite prepared from a metal-organic framework (type UiO-67) and graphene.

An aptamer based assay is described for the determination of Salmonella typhimurium (S.typhimurium). A metal-organic framework-graphene composite of type UiO-67/GR is used as the substrate, and an aptamer-gold nanoparticles-horseradish peroxidase (Apt-AuNP-HRP) conjugate the signal amplification probe. A phosphate-terminal and partially complementary DNA (cDNA) of the aptamer is covalently bound to UiO-67/GR via the chemical complexation between phosphate and Zr-OH groups of UiO-67, and then S. typhimurium and cDNA will compete for the binding sites. The binding of Apt-AuNP-HRP to S.typhimurium leads to the formation of strong conjugates. The unbound signal probes then attach to the surface of a glassy carbon electrode via hybridization with cDNA. This generates a large current response (best measured at a potential as low as -0.02V vs. saturated calomel electrode) under the catalytic action of HRP on the H2O2-hydroquinone system. Under the optimal conditions, the differential pulse voltammetric signal decreases linearly in the 2 × 101 -2 × 108cfu·mL-1S.typhimurium concentration range, with a lower detection limit of 5cfu·mL-1 (based on S/N = 3). The method was successfully applied to the detection of S. typhimurium in spiked milk samples. Graphical abstract Schematic presentation of electrochemical determination of Salmonella typhimurium (S.typhimurium). A metal-organic framework (type UiO-67) and graphene (GR) composite were used as substrate, and gold nanoparticles carrying horseradish peroxidase (HRP) for signal amplification. HQ: hydroquinone; cDNA: complementary DNA of aptamer.

Han Chinese family with early-onset Parkinson's disease carries novel compound heterozygous mutations in the PARK2 gene.

PurposeTo identify deletions, duplications, and point mutations in 55 previously reported genes associated with Parkinson's disease (PD) and certain genes associated with tremor, spinocerebellar ataxia, and dystonia in a Han Chinese pedigree with early‐onset Parkinson's disease (EOPD).Patients and MethodsClinical examinations and genomic analyses were performed on six subjects belonging to three generations of a Han Chinese family. Target region capture and high‐throughput sequencing were used to screen these genes associated with PD, tremor, spinocerebellar ataxia, and dystonia. The multiplex ligation‐dependent probe amplification (MLPA) method was applied to detect rearrangements in PARK2 exons. Direct Sanger sequencing of samples from all subjects further verified the detected abnormal PRKRA, SPTBN2, and ATXN2 gene fragments.ResultsTwo family members were diagnosed with PD based on the clinical manifestations, imaging analyses. PARK2 gene heterozygous deletion of exon 3 and heterozygous duplication of exon 6 were identified in them (II‐3 and 4). A single heterozygous deletion of exon 3 in PARK2 was detected in II‐5 and III‐10. A single duplication of exon 6 in PARK2 was detected in I1. Both the heterozygous mutation c.2834G>A (p. R945H) in exon 16 and the heterozygous mutation c.1924 C>T (p. R642W) in exon 14 of the SPTBN2 gene were identified in II‐3, II‐4, and III‐10. The heterozygous mutation c.2989 C>T (p. R997X) in exon 24 of the ATXN2 gene was detected in II‐4 and II‐5, and the heterozygous mutation c.170 C>A (p. S57Y) in exon 2 of the PRKRA gene was detected in II‐3, II‐4, and III‐10. Other mutations in some genes associated with PD, tremor, spinocerebellar ataxia, and dystonia were not detected.ConclusionsNovel compound heterozygous mutations were identified in a Han Chinese pedigree and might represent a cause of EOPD.

Direct and sensitive detection of circulating miRNA in human serum by ligase-mediated amplification

MicroRNAs (miRNA) involve in regulating different physiological processes whose dysregulation is associated with a wide range of diseases including cancers, diabetes and cardiovascular problems. Herein, we report a direct, sensitive and highly selective detection assay for circulating microRNA (miRNA). This detection strategy employs magnetic nanoparticles as the reaction platform which can not only allow online pre-concentration and selective separation but also integrates ligation reaction with amplification to enhance the sensitivity of the detection assay. With the presence of the target miRNA, the locked nucleic acid (LNA)-modified molecular beacon (MB) opens up, exposing the binding sites at two ends. The 3’- and 5’-end of the MB responsible for the attachment onto the magnetic nanoparticles, and reporting probe for the attachment of the pair of amplification probes respectively. The ligase ligate RNA to DNA enhance the amplification efficiency. Upon labelled with intercalating fluorophores (YOYO-1) on the hybrids, the quantification of the target miRNA was determined by measuring the fluorescence intensity. A detection limit of 314 fM was achieved with trace amount of sample consumption (~20 μL). As a proof of concept, miRNA-149 was chosen as the target miRNA. This assay is capable of discriminating single-base and reliably quantifying circulating miRNA-149 in both healthy and cancer patient’s serums. The result obtained was comparable with that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), suggesting that this direct and sensitive assay can be served as a promising, non-invasive tool for early diagnosis of breast cancer and colorectal cancer.

Aptamer-Functionalized and Gold Nanoparticle Array-Decorated Magnetic Graphene Nanosheets Enable Multiplexed and Sensitive Electrochemical Detection of Rare Circulating Tumor Cells in Whole Blood.

The identification and monitoring of circulating tumor cells (CTCs) in human blood plays a pivotal role in the convenient diagnosis of different cancers. However, it remains a major challenge to monitor these CTCs because of their extremely low abundance in human blood. Here, we describe the synthesis of a new aptamer-functionalized and gold nanoparticle (AuNP) array-decorated magnetic graphene nanosheet recognition probe to capture and isolate rare CTCs from human whole blood. In addition, by employing the aptamer/electroactive species-loaded AuNP signal amplification probes, multiplexed electrochemical detection of these low levels of CTCs can be realized. The incubation of the probes with the sample solutions containing the target CTCs can lead to the efficient separation of the CTCs and result in the generation of two distinct voltammetric peaks on a screen-printed carbon electrode, whose potentials and current intensities, respectively, reflect the identity and number of CTCs for the multiplexed detection of the Ramos and CCRF-CEM cells with detection limits down to 4 and 3 cells mL-1. With the successful demonstration of the concept, further extension of the developed sensing strategy for the determination of various CTCs in human whole blood for the screening of different cancers can be envisioned in the near future.

DNA synergistic enzyme-mediated cascade reaction for homogeneous electrochemical bioassay

Enzyme-mediated cascade reaction is applied to amplify signal and decrease the background because enzyme can catalyze inactive substrates into active substrates to generate the signal. In this work, Au nanoparticles, as signal probe, are used to load DNA probe and ALP for dual signal amplification. Based on enzyme-mediated cascade reaction, a homogeneous biosensor is constructed for bioassay by employing thrombin as target molecule. When the target is present in the solution, ALP catalyzes the PPi into Pi and then reacts with molybdate in conjunction with Pi in the DNA backbone to produce redox precipitates on the surface of the reduced graphene oxide modified electrode with the help of magnetic separation. Compared with the conventional heterogeneous biosensor, the immobilization-free strategy, proposed in this homogeneous biosensor, improves the sensitivity because of its lower steric hindrance. As a result, this biosensor displayed a great sensitivity with a wide linear range from 1 fM to 10 nM and a detection limit of 0.26 fM, providing a promise and easy operating method for various proteins detection.

A sensitive and specific real-time PCR targeting DNA from wheat, barley and rye to track gluten contamination in marketed foods

This work reports a real-time PCR assay to specifically detect the presence of DNA from the main gluten-containing cereals wheat, barley and rye (WBR) in foodstuffs. The WBR real-time PCR uses two specific primers and a Taqman probe for amplification of a 118 bp DNA fragment common to the three target cereals on the ribosomal internal transcribed spacer (ITS) region. In-house validation of the method was performed employing three binary arrays containing different concentrations (100 000–10 mg/kg) of a wheat/barley/rye mixture in maize flour: untreated, soft heat-treated at 160 °C/13 min and intense heat-treated at 200 °C/20 min. Results showed optimal PCR amplification efficiency, reproducibility and sensitivity with an overall limit of detection of 10–50 mg/kg of the target cereals, theoretically equating to approximately 1–5 mg/kg of gluten. Applicability of the assay was further assessed through a market analysis of 220 food products by the real-time PCR assay and the official R5-based ELISA. Comparative results highlighted the ability of the proposed methodology as a highly sensitive and robust procedure to detect the presence of potentially harmful concentrations of undeclared gluten-containing cereals in some of the tested samples, supporting results of the immunological assays currently used for gluten determination in foods.