Amplification Of Target Genes Research Articles

Screening of strictocidine synthase and lysine decarboxylase gene involved in alkaloid biosynthesis in plants of Catharanthus rosea, Adathoda vasica and Wedelia trilobata

Alkaloids are naturally occurring nitrogenous organic compounds of plant and display vast range of pharmacological properties such as anticancer, antiviral, anti-diabetic, antioxidant and antimicrobial activities. Medicinal plants are the main source of secondary metabolites and the enzymatic pathway involved in its production may be enhanced to increase synthesis of metabolites. The strictocidine synthase (STR) and lysine decarboxylase (LDC) gene are involved in biosynthesis of monoterpenoid indole alkaloid and quinolizidine alkaloid respectively. In the present study, STR and LDC gene were analyzed in Catharanthus roseus, Adathoda vasica and Wedelia trilobata using real time PCR. The PCR results showed S shaped curve with Ct value for amplification of target gene which was found in Catharanthus roseus for STR gene and in Catharanthus roseus and Adathoda vasica for LDC gene. For further confirmation of target, gene samples were separated on agarose gel for size determination and the size of STR gene was found to be 1.1 kb and LDC gene was 1.3 kb respectively. Thus this study should be beneficial for academic as well as industrial point of view from production of alkaloids in plants by enhancing STR and LDC gene synthesis and alkaloids should also be used for isolation and characterization for pharmaceutical applications.

Efficacy and Safety of Nasal Immunisation with Somatostatin DNA Vaccine for Growth Promotion in Fattening Pigs.

This study aimed to evaluate the efficacy and safety of the SS DNA vaccine on growing pigs. Randomly, 147 pigs were divided into four groups, treatment 1 (T1, 3 × 109 CFU/mL, n = 39), T2 (3 × 108 CFU/mL, n = 35), T3 (3 × 107 CFU/mL, n = 35) and control group (phosphate-buffered saline, n = 38). All animals received two vaccinations separated by 45 days and the same diet and management. The results showed that all treatment groups (T1, T2 and T3) had significantly higher slaughter weight (d 185) than the Ctrl group (p < 0.05), and daily gain between 50 and 110 days of age was significantly higher in all treatment groups than in the Ctrl group (p < 0.05). Antibody-positive pigs have significantly higher daily weight gain than that in antibody-negative pigs (p < 0.05). The results of the meat quality analysis showed no significant changes between the P (antibody-positive pigs) and N (antibody-negative pigs) groups. Furthermore, the results showed that antibody titres at 110 and 185 days had a significant positive correlation with the daily weight gain (p < 0.05) and a significant negative correlation with the backfat thickness (p < 0.05). Evaluating the safety of vaccines by PCR amplification of target genes (GS/2SS), faecal, soil and water samples had no target genes detected by PCR amplification in these samples after 5 days, and no GS/2SS were detected in the blood and tissues for the experimental period. Moreover, no abnormalities were found in pathological sections of the P group compared with the N group. In conclusion, SS DNA vaccines can promote the growth of fattening pigs to a certain extent without altering the meat quality, and it has no effects on the safety of the surrounding environment.

High sensitivity SARS-CoV-2 detection using graphene oxide-multiplex qPCR

The emerging pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) critically challenges early and accurate virus diagnoses. However, the current gold standard for SARS-CoV-2 detection, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), has reportedly failed to detect low-viral loads. One compound, graphene oxide (GO), which adsorbs single-stranded DNA (ssDNA), has been widely applied in molecular pathogen detection. This study presents a highly sensitive GO-multiplex qPCR method for simultaneous detection of two SARS-CoV-2 genes (RdRP and E) and one reference gene (RNase P). In a GO-multiplex qPCR system, GO pre-absorbs each forward primer to form specific GO-forward primer composites before entering the amplification system. Target gene amplification is confined within the primer-enriched composites, thus, improving the sensitivity of the assay. Compared to conventional multiplex qPCR, GO-multiplex qPCR reduces the limit of detection by 10-fold to 10 copies/reaction. Hence, the GO-multiplex qPCR assay can be effectively used for SARS-CoV-2 detection.

Near infrared light-induced dynamic modulation of enzymatic activity through polyphenol-functionalized liquid metal nanodroplets

Dynamic manipulation of enzymatic activity is a challenging task for applications in chemical and pharmaceutical industries due to the difficult modification and variable conformation of various enzymes. Here, we report a new strategy for reversible dynamic modulation of enzymatic activity by near-infrared light-induced photothermal conversion based on polyphenol-functionalized liquid metal nanodroplets (LM). The metal-phenolic nanocoating not only provides colloidal stability of LM nanodroplets but also generates nanointerfaces for the assembly of various enzymes on the LM nanodroplets. Upon near infrared (NIR) irradiation, the localized microenvironmental heating through photothermal effect of the LM nanodroplets allows tailoring the enzymatic activity without affecting the bulk temperature. A library of functional enzymes, including proteinase K, glucoamylase, glucose oxidase, and Bst DNA polymerase, is integrated to perform a reversible control and enhanced activities even after five times of cycles, demonstrating great potential in bacterial fermentation, bacteriostasis, and target gene amplification.

Morphological and Molecular Identification of Oestrus ovis (Diptera: Oestridae) Larvae Collected from a Chinese Patient with Conjunctival Myiasis.

Human conjunctival myiasis, which is often misdiagnosed or missed clinically, is commonly caused by Oestrus ovis larvae. Here, pathogenic identification was performed for two maggots collected from a patient from China, to provide a clinical scientific basis for diagnosis and treatment. Morphological identification was performed using a microscope. Oestrus mtDNA cox1 and rDNA 28S were selected as target genes for duplex PCR amplification, followed by cloning, sequencing, and identification. Morphological examination showed that the maggots were approximately 1.0-1.5mm long, long-oval-shaped, segmented, and covered with small spines, with a pair of hooks in the scolex and claw-like spines at the telson. Therefore, they were identified as the first-instar larvae of O. ovis. Duplex PCR detected products of approximately 400 and 200bp, consistent with the size of designed cox1 and 28S D7a gene fragments, respectively. Sequences of cox1 and 28S D7a from the samples in question had 99.5-100.0% and 96.2-100.0% similarity (respectively) to GenBank sequences of O. ovis specimens known to parasitize sheep, goats, and humans. However, some 28S D7a sequences exhibited 89.7-90.6% similarity to GenBank sequences of Oestrus sp. known to parasitize Capra pyrenaica (Artiodactyla: Bovidae) (Iberian ibex). Therefore, we considered that the larvae infecting the patient originated from sheep or goats, not Iberian ibex. The phylogenetic trees supported this conclusion. This study implemented the first duplex PCR molecular identification of O. ovis larvae parasitizing human eyes in China as a complementary approach to morphological identification. Our results indicate that molecular tools can be utilized to aid in the diagnosis of opthalmomyiasis.

Modulating gene regulation function by chemically controlled transcription factor clustering

Recent studies have suggested that transcriptional protein condensates (or clusters) may play key roles in gene regulation and cell fate determination. However, it remains largely unclear how the gene regulation function is quantitatively tuned by transcription factor (TF) clustering and whether TF clustering may confer emergent behaviors as in cell fate control systems. Here, to address this, we construct synthetic TFs whose clustering behavior can be chemically controlled. Through single-parameter tuning of the system (i.e., TF clustering propensity), we provide lines of evidence supporting the direct transcriptional activation and amplification of target genes by TF clustering. Single-gene imaging suggests that such amplification results from the modulation of transcriptional dynamics. Importantly, TF clustering propensity modulates the gene regulation function by significantly tuning the effective TF binding affinity and to a lesser extent the ultrasensitivity, contributing to bimodality and sustained response behavior that are reminiscent of canonical cell fate control systems. Collectively, these results demonstrate that TF clustering can modulate the gene regulation function to enable emergent behaviors, and highlight the potential applications of chemically controlled protein clustering.

Pan-cancer analysis of mutations in open chromatin regions and their possible association with cancer pathogenesis.

BackgroundOpen chromatin is associated with gene transcription. Previous studies have shown that the density of mutations in open chromatin regions is lower than that in flanking regions because of the higher accessibility of DNA repair machinery. However, in several cancer types, open chromatin regions show an increased local density of mutations in activated regulatory regions. Although the mutation distribution within open chromatin regions in cancer cells has been investigated, only few studies have focused on their functional implications in cancer. To reveal the impact of highly mutated open chromatin regions on cancer, we investigated the association between mutations in open chromatin regions and their possible functions.MethodsWhole‐genome sequencing data of 18 cancer types were downloaded from the PanCancer Analysis of Whole Genomes and Catalog of Somatic Mutations in Cancer. We quantified the mutations located in open chromatin regions defined by The Cancer Genome Atlas and classified open chromatin regions into three categories based on the number of mutations. Then, we investigated the chromatin state, amplification, and possible target genes of the open chromatin regions with a high number of mutations. We also analyzed the association between the number of mutations in open chromatin regions and patient prognosis.ResultsIn some cancer types, the proportion of promoter or enhancer chromatin state in open chromatin regions with a high number of mutations was significantly higher than that in the regions with a low number of mutations. The possible target genes of open chromatin regions with a high number of mutations were more strongly associated with cancer than those of other open chromatin regions. Moreover, a high number of mutations in open chromatin regions was significantly associated with a poor prognosis in some cancer types.ConclusionsThese results suggest that highly mutated open chromatin regions play an important role in cancer pathogenesis and can be effectively used to predict patient prognosis.

Insertion Mutation of MSMEG_0392 Play an Important Role in Resistance of M. smegmatis to Mycobacteriophage SWU1.

PurposePhage is a new choice for the treatment of multi-drug-resistant bacteria, and phage resistance is also an issue of concern. SWU1 is a mycobacteriophage, and the mechanism of its resistance remain poorly understood.MethodsThe mutant strains which were stably resistant to SWU1 were screened by transposon mutation library. The stage of phage resistance was observed by transmission electron microscope (TEM). The insertion site of transposon was identified by thermal asymmetric interlaced PCR (TAIL-PCR). The possible relationship between insertion site and phage resistance was verified by gene knockout technique. The fatty acid composition of bacterial cell wall was analyzed by Gas Chromatography-Mass Spectrometer (GC-MS). Through the amplification and sequencing of target genes and gene complement techniques to find the mechanism of SWU1 resistance.ResultsThe transposon mutant M12 which was stably resistant to mycobacteriophage SWU1 was successfully screened. It was confirmed that resistance occurred in the adsorption stage of bacteriophage. It was verified that the insertion site of the transposon was located in the MSMEG_3705 gene, but after knocking out the gene in the wild type M. smegmatis mc2 155, the resistance of the knockout strain to SWU1 was not observed. Through the amplification and sequencing of the target gene MSMEG_0392, it was found that there was an adenine insertion mutation at position 817. After complementing MSMEG_0392 in M12, it was found that M12 returned to sensitivity to SWU1.ConclusionWe confirmed that the resistance of M12 to SWU1 was related to the functional inactivation of MSMEG_0392 and this phenomenon may be caused by the change of cell wall of M. smegmatis.

A continuous flow PCR array microfluidic chip applied for simultaneous amplification of target genes of periodontal pathogens.

The concept of time to place conversion makes using a continuous flow polymerase chain reaction (CF-PCR) microfluidic chip an ideal way to reduce the time required for amplification of target genes; however, it also brings about low throughput amplicons. Although multiplex PCR can simultaneously amplify more than one target gene in the chip, it may easily induce false positives because of cross-reactions. To circumvent this problem, we herein fabricated a microfluidic system based on a CF-PCR array microfluidic chip. By dividing the chip into three parts, we successfully amplified target genes of Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f) and Treponema denticola (T.d). The results demonstrated that the minimum amplification time required for P.g, T.d and T.f was 2'07'', 2'51'' and 5'32'', respectively. The target genes of P.g, T.d and T.f can be simultaneously amplified in less than 8'05''. Such a work may provide a clue to the development of a high throughput CF-PCR microfluidic system, which is crucial for point of care testing for simultaneous detection of various pathogens.

Retroreflection-based sandwich type affinity sensing of isothermal gene amplification products for foodborne pathogen detection.

Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics, as the rapid, specific, and sensitive amplification of target genes is possible. However, it is necessary to measure fluorescence in the quantitative analysis of LAMP products, so a sophisticated optical setup is required. This study tried to develop a novel sensing method that can quantify target analytes with simple equipment, such as nonspectroscopic white light and a CMOS camera. To achieve this, a retroreflective Janus particle (RJP) as a probe and specially designed loop primers, fluorescein isothiocyanate (FITC)- and biotin-modified loop primers, were introduced into the LAMP system. By performing LAMP in the presence of designed primers, double-stranded amplicons possessing FITC and biotin labels at each end are generated in proportion to the quantity of the target pathogen. Using the anti-FITC antibody-modified sensing surface and streptavidin-conjugated RJP probes, the amplicons can be captured in sandwich-configuration and detected under nonspectroscopic conditions composed of white light and a camera. To confirm the feasibility of the sensing system, the invA gene of Salmonella was selected as the target. It was possible to quantitatively analyze the Salmonella concentration from 0 to 106 colony-forming units, sufficiently covering the required detection range. In addition, quantitative analyses of pathogens in contaminated food sources, including milk and chicken meat, were successfully conducted with a limit of detection of 10 CFU.

Novel CRISPR/Cas12a-based genetic diagnostic approach for SLC26A4 mutation-related hereditary hearing loss

Hereditary hearing loss is a common defect of the auditory nervous system with high-incidence, seriously affecting the quality of life of the patients. The clinical manifestations of SLC26A4 mutation-related hearing loss are congenital sensorineural or mixed deafness. Sensitive and specific SLC26A4 mutation detection in the early clinical stage is key for the early indication of potential hearing loss in the lack of effective treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology, we designed a fast and sensitive detection system for SLC26A4 pathogenic mutations (c.919-2A > G, c.2168A > G and c.1229C > T). This recombinase-aided amplification-based detection system allows rapid target gene amplification and, in combination with the CRISPR-based nucleic acid testing (NAT) system, mutation site detection. Moreover, mismatches were introduced in CRISPR-derived RNA (crRNA) to increase signal differences between the wild-type genes and mutant genes. A total of 64 samples were examined using this approach and all results were verified using Sanger sequencing. The detection results were consistent with the polymerase chain reaction-Sanger sequencing results. Overall, this CRISPR-based NAT technology provides a sensitive and fast new approach for the detection of hereditary deafness and provides a crRNA optimization strategy for single-nucleotide polymorphism detection, which could be helpful for the clinical diagnosis of SLC26A4 mutation-related hereditary hearing loss.

Development of a nucleic acid chromatography assay for the detection of commonly isolated rapidly growing mycobacteria.

Introduction. Non-tuberculosis mycobacterium infections are increasing worldwide, including those caused by rapidly growing mycobacteria (RGM).Gap Statement. The identification of the aetiological agent in the context of infections is essential for the adoption of an adequate therapeutic approach. However, the methods for the rapid distinction of different RGM species are less than optimal.Aim. To develop a nucleic acid chromatography kit to identify clinically common RGM.Methodology. We tried to develop a nucleic acid chromatography kit designed to detect four RGM species (including three subspecies) i.e. Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bolletii (detected as M. abscessus/bolletii) Mycobacterium abscessus subsp. massiliense, Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium peregrinum. The amplified target genes for each species/subspecies using multiplex PCR were analysed using a nucleic acid chromatography assay.Results. Among the 159 mycobacterial type strains and 70 RGM clinical isolates tested, the developed assay correctly identified all relevant RGM without any cross-reactivity or false-negatives. The limits of detection for each species were approximately 0.2 pg µl-1.Conclusion. The rapid and simple nucleic acid chromatography method developed here, which does not involve heat denaturation, may contribute to the rapid identification and treatment of RGM infections.

Molecular characterization of intraspecific variations in Helicoverpa armigera (Hübner) populations across India

Aim: The present study was undertaken to explore the genetic diversity among Helicoverpa armigera populations from varied geographic regions of India using mitochondrial cytochrome c oxidase I (COI) gene fragments. Methodology: The larval specimens of H. armigera collected from 20 locations were subjected to DNA extraction, PCR amplification of target gene, sequencing and then multiple sequence alignments. Results: Based on COI sequence data, high levels of genetic differentiation among some H. armigera populations were detected, but divergence existing was not high enough to delineate them as separate species. The Indian population as a whole exhibited similarity with global genetic assemblage. Significant negative neutrality test indices and unimodal mismatch distribution further supported that this insect experienced a demographic expansion in the past. The phylogenetic tree and median-joining haplotype network indicated that genetic similarity was not related with geographic proximity of populations. Interpretation: Differences based on genetic analyses indicate considerable subspecific level variations among H. armigera populations of India. However, there is no existence of any unidentified cryptic species of H. armigera in the country.

Spectrum and frequency of connexin 26 & connexin 30 gene mutations in patients with congenital hearing loss from Ladakh India

BackgroundCongenital hearing loss refers to prenatal hearing impairment, which is the commonly shared problem among sensory disorders that ranges from slight to profound. In this study, we screened the involvement of connexin 26 & connexin 30 gene in congenital hearing loss patients from the Kargil district of Ladakh, India by target gene sequencing. Materials and methodsThe peripheral blood sample of the patients was collected followed by DNA extraction and PCR of the target gene of interest. The amplified target gene was then sequenced and analysed for any changes in nucleotide sequence. ResultsIn this study, the connexin 26 & connexin 30 are the target gene that are associated with hearing loss and in this study, we found mutations in the connexin 26 gene and none of the mutations were found in the connexin 30 gene. The mutations found in the connexin 26 gene are “NM_004004:c.71G>A, “NM_004004:c.223C>T, “NM_004004:c230G>A, “NM_004004:c.380G>A, and “NM_004004:c.439G>A which change the amino acids to W24X, R75W, W77X, R127H, and E147K respectively and the frequency of the connexin 26 mutations in patients was 23% and the overall mutations in the cohort were 14.5%. This study found that “NM_004004:c.71G>A (W24X) nucleotide variant as common mutation. Both homozygous as well as heterozygous variants were found. R127H was the only mutation observed in control samples. A significant association was seen between connexin 26 gene mutations and familial history of congenital hearing loss and consanguinity with the p-value P<0.001. Conclusion and significanceAlthough the etiology has been found to be multifactorial. However, the role of any nucleotide variant in the gene of interest needs to be thoroughly investigated to study the genetic etiology of the anomalies. The results of this study showed a positive association between congenital hearing loss and the connexin 26 gene. However, we did not find any role of the connexin 30 gene in congenital hearing loss.

A sampling survey of enterococci within pasteurized, fermented dairy products and their virulence and antibiotic resistance properties.

Globally, fermented foods (FFs), which may be traditional or industrially-produced, are major sources of nutrition. In the traditional practice, the fermentation process is driven by communities of virtually uncharacterized microflora indigenous to the food substrate. Some of these flora can have virulent or antibiotic resistance properties, posing risk to consumers. Others, such as Enterococcus faecalis and Enterococcus faecium, may also be found in such foods. Enterococci that harbor antibiotic resistance or virulence factors can cycle among animals, food, humans and the environment, thereby transferring these harmful properties at the gene level to harmless commensals in the food matrix, animals and humans. In this work, several microbial isolates obtained from different FF sources were analyzed for their identity and virulence and/or antibiotic resistance properties. For identification aiming at enterococci, isolates that were Gram-positive and catalase- and oxidase-negative were subjected to multiple tests including for growth in broth containing 6.5% NaCl, growth and hydrolytic activity on medium containing bile-esculin, hemolytic activity on blood agar, and growth at 45°C and survival after incubation at 60°C for 30 min. Furthermore, the isolates were tested for susceptibility/resistance to a select group of antibiotics. Finally, the isolates were molecularly-characterized with respect to species identity and presence of virulence-encoding genes by amplification of target genes. Most sources contained enterococci, in addition to most of them also containing Gram-negative flora. Most of these also harbored virulence factors. Several isolates were also antibiotic-resistant. These results strongly suggest attention should be given to better control presence of such potentially pathogenic species.

Primer evaluation and development of a droplet digital PCR protocol targeting amoA genes for the quantification of Comammox in lakes

To date, little is known about the ecological significance of Comammox (COMplete AMMonia OXidizers) Nitrospira in the water column of freshwater lakes. Water samples collected along depth profiles were used to investigate the distribution of Comammox in 13 lakes characterized by a wide range of physicochemical properties. Several published primers, which target the α-subunit of the ammonia monooxygenase, generated non-specific PCR products or did not amplify target genes from lake water and other habitats. Therefore, a new primer set has been designed for specific detection of Comammox in lakes. The high specificity of the PCR assay was confirmed by sequencing analysis. Quantification of Comammox amoA genes in lake water samples based on droplet digital PCR (ddPCR) revealed very low abundances (not exceeding 85 amoA copies ml−1), which suggest that Comammox is of minor importance for the nitrification process in the water column of the study sites. Surprisingly, samples taken from the sediment/water-interface along an oxygen gradient in dimictic Piburger See showed Comammox abundances three to four magnitudes higher than in the pelagic realm of the lake, which indicates a preference of Comammox to a particle-attached lifestyle.

Rapid and highly sensitive pathogen detection by real-time DNA monitoring using a nanogap impedimetric sensor with recombinase polymerase amplification

Fast detection of pathogens is important for protecting our health and society. Herein, we present a high-performance nanogap impedimetric sensor for monitoring nucleic acid amplification in real time using isothermal recombinase polymerase amplification (RPA) for rapid pathogen detection. The nanogap electrode chip has two pairs of opposing gold electrodes with a 100 nm gap and was fixed to a PCB. Then, the nanogap impedimetric sensor was immersed in RPA reaction solution for the detection of E. coli O157:H7, and target DNA amplification was evaluated through bulk solution impedance changes using impedance spectroscopy every minute during RPA. In addition, target gene amplification in the sample solution during RPA was confirmed with a 2% DNA agarose gel. Our nanogap impedimetric sensor can detect down to a single copy of the eae A gene in gDNA extracted from E. coli O157:H7 as well as a single cell of pathogenic E. coli O157:H7 strain within 5 min during direct RPA, which was performed with the pathogen itself and without the extraction and purification of target gDNA. The miniaturized nanogap impedimetric sensor has potential as a cost-effective point-of-care device for fast and accurate portable pathogen detection via real-time nucleic acid analysis.

Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

BackgroundThe development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT).ResultsWestern blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT.ConclusionOur findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

Multiplex amplification of target genes of periodontal pathogens in continuous flow PCR microfluidic chip.

Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f) are believed to be the major periodontal pathogens that cause gingivitis, which affects 50-90% of adults worldwide. Microfluidic chips based on continuous flow PCR (CF-PCR) are an ideal alternative to a traditional thermal cycler, because it can effectively reduce the time needed for temperature transformation. Herein, we explored multi-PCR of P.g, T.d and T.f using a CF-PCR microfluidic chip for the first time. Through a series of experiments, we obtained two optimal combinations of primers that are suitable for performing multi-PCR on these three periodontal pathogens, with amplicon sizes of (197 bp, 316 bp, 226 bp) and (197 bp, 316 bp, 641 bp), respectively. The results also demonstrated that by using multi-PCR, the amplification time can be reduced to as short as 3'48'' for the short-sized amplicons, while for T.f (641 bp), the minimum time required was 8'25''. This work provides an effective way to simultaneously amplify the target genes of P.g, T.d and T.f within a short time, and may promote CF-PCR as a practical tool for point-of-care testing of gingivitis.

Fullerenol as a photoelectrochemical nanoprobe for discrimination and ultrasensitive detection of amplification-free single-stranded DNA

Traditional approaches for nucleic acids detection require prior amplification of target genes, while nanomaterials-aided DNA biosensors are very magnificent but still suffer from the nanomaterial acquirement and limited sensitivity (above picomolar level). Herein, fullerenol C60(OH)25, a representative fullerene derivative, was employed as a photoelectrochemical (PEC) nanoprobe to achieve discrimination and ultrasensitive detection of amplification-free single-stranded DNA (ssDNA) down to sub-femtomolar level. The bonded hydroxyl groups with intense density endowed fullerenol to directly recognize and capture ssDNA-AuNPs via the hydrogen bonding interactions (H-bonds), leading to a sharply decreased photocurrent with quenching efficiency up to 85%, which could be attributed to the photo-generated electrons on the conduction band of fullerenol (−4.66 eV) preferentially migrating to the Fermi level of AuNPs (−5.1 eV) rather than the electrode. In the presence of target gene (mutant human p53 gene fragment), the H-bonds between fullerenol and ssDNA were competitively depleted during the base pairing process of complete hybridization between ssDNA and target, making double-stranded DNA-AuNPs (dsDNA-AuNPs) depart so that the photocurrent powerfully recovered. On basis of the photocurrent variation before and after target introduction, this proposed simple, rapid and ultrasensitive PEC biosensor for amplification-free target gene detection illustrated a wide liner ranged from 1 fM to 100 pM and a detection limit of 0.338 fM. This work presented an ingenious strategy for the discrimination and ultrasensitive detection of nucleic acids, and the well-designed PEC biosensor was further conducive to the impetus of clinic diagnostics.