An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques

The neglected tropical disease mycetoma can become extremely devastating, and can be caused both by fungi and bacteria; these are popularly known as eumycetoma and actinomycetoma respectively. The classical triad of the disease is subcutaneous swelling, multiple discharging sinuses and the presence of macroscopic granules. The present study aims to highlight the existing diagnostic modalities and the need to incorporate newer and more advanced laboratory techniques like pan fungal/pan bacterial 16S rRNA gene polymerase chain reaction (PCR) and sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). It is important for the medical team to be aware of the various diagnostic options (both existing and future), so that diagnosis of such a debilitating disease is never missed, both by clinicians and microbiologists/pathologists. The newer diagnostic methods discussed in this article will help in rapid, accurate diagnosis thus facilitating early treatment initiation, and decreasing the overall morbidity of the disease. In the Indian context, newer technologies need to be made available more widely. Making clinicians aware and promoting research and development in mycetoma diagnostics is the need of the hour.

Molecular tandem repeat strategy for production ofultrashort peptides / 生物工程学报

Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)n. The pET28a-Trxm-(TRSP)n was introduced into E. coli BL21(DE3) to generate a library of Trxm-(TRSP)n sequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)1 achieved 50% of the total protein, while the expression level of Trxm-(TRSP)2 was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)1 were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.

Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations / 生物工程学报

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

Coating with flexible DNA network enhanced T-cell activation and tumor killing for adoptive cell therapy

Adoptive cell therapy (ACT) is an emerging powerful cancer immunotherapy, which includes a complex process of genetic modification, stimulation and expansion. During these

Progress in rolling circle amplification in biological detection / 生物工程学报

Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.

A DNA polyaptamer system as a targeted antitumor drug delivery / 中国药科大学学报

@#A long chain structure of DNA(polyaptamer)composed of multiple aptamer units was synthesized by rolling circle amplification and used for the construction of polyaptamer-doxorubicin system in the treatment of leukemia cells. It was found that the system was significantly more effective than monoaptamer in targeting and killing leukemia cells as it provided 35-fold enhanced binding affinity and 10-fold greater drug loading via multivalent effects. Drug release and cell viability also proved that the conjugates could gain entrance into the cells and rapidly release doxorubicin under the action of lysosome, leading to the tumoricidal effect.

Current progress in quantitative detection of microRNA / 中国药科大学学报

@#With the recent development of investigating biological functions of microRNA(miRNA), the importance of miRNA in the biological processes including developmental biology, metabolic regulation, disease progression and treatment has been well recognized. Due to its the instability, low level of expression and minor difference in sequence, more sophisticated analytical methods for rapid and easy quantitative detection of miRNA with strong specificity and high sensitivity play an important role in the study of the biological functions of miRNA. This review analyzes and compares recent quantitative methods to detect miRNA, with an attempt to provide a foundation for choosing an appropriate method to quantitatively analyze miRNA.

Highly Sensitive Fluorescent Aptasensor for Thrombin Detection Based on Competition Triggered Rolling Circle Amplification / 分析化学

Based on the competition reaction of target protein, aptamer probe, padlock probe and complementary sequence, a highly sensitive fluorescent aptasensor was developed in this study in combination with rolling circle amplification. In the absence of target protein, the ligation-rolling circle amplification reaction was repressed because the complementary sequence hybridized with aptamer probe to form double-stranded duplex. While in the presence of target protein, the target molecules bound specifically with aptamer probe, inducing displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of E. coli DNA ligase, and the rolling circle amplification process could be accomplished by Phi 29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacon ( the detection probes) , resulting in a significant fluorescence signal. The effects of length of complementary DNA ( CDNA ) sequence and concentration of padlock probe were investigated. Under the optimized experimental conditions, the model target protein thrombin could be highly sensitively detected by the proposed aptasensing system in a linear range of 0 . 067-32 . 4 nmol/L with a detection limit of 0 . 03 nmol/L ( approximately 90 amol target molecules). Moreover, the presented sensing method was universal for other target analysis by skillfully design of the sequence of aptamer probe and related oligonucleotides.

Concurrent Infection of Torque Teno Sus Virus and Porcine Circovirus Type 2 in a Clinical Case with Post-weaning Multisystemic Wasting Syndrome.

Aims: The goal of this study was to identify possible concurrent infection of torque teno sus virus (TTSuV) and porcine circovirus type 2 (PCV2) in a clinical case with postweaning multisystemic wasting syndrome (PMWS) on certain farm of Shanghai, China. Place and Duration of Study: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, between June 2009 and June 2010 & Institute of Animal Health, Guangdong Academy of Agricultural Sciences, between September and November, 2013. Methodology: Multiply-primed rolling-circle amplification (MPRCA), a useful molecular tool, was performed to amplify genome sequence of TTSuV and PCV2. For serum sample of SH0822 from a clinical case with PMWS, the products of MPRCA were digested using EcoR I, Xba I, Sma I, Sac I, respectively. Moreover, Clustal W program (DNASTAR software) and MEGA 5.1 software (neighbour-joining method) was used to analysis its nucleotide homology and genetic relationship. Results: Restriction digestion analysis showed one TTSuV genome-size fragment was presented in 1.2 % agarose gel, moreover, another PCV2 genome-size fragment was also presented. Nucleotide sequencing and phylogenetic analysis results suggested that its complete genome were 2823-nucleotide size and 1767-nucleotide size and they were divided into species TTSuV1b and genotype PCV2b, respectively. Conclusion: Concurrent infection of TTSuV and PCV2 in a clinical case with PMWS was identified using MPRCA combining with restriction endonuclease digestion, which indicated that MPRCA was an effective tool to attain simultaneous detection and genome amplification of TTSuV and PCV2.

A Fosmid Cloning Strategy for Detecting the Widest Possible Spectrum of Microbes from the International Space Station Drinking Water System

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

Highly sensitive chemiluminescence technology for protein detection using aptamer-based rolling circle amplification platform / 药物分析学报

A robust,selective and highly sensitive chemiluminescent (CL) platform for protein assay was presented in this paper.This novel CL approach utilized rolling circle amplification (RCA) as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier.Typically,the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer-primer sequence.This aptamer-primer sequence was then employed as the primer of RCA.Based on this design,a number of the biotinylated probes and streptavidin-horseradish peroxidase (SA-HRP) were captured on the plate,and the CL signal was amplified.In summary,our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized,and devoid of light source.Therefore,this new technique will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases,by taking advantages of high sensitivity and selectivity.

Detection of TAC1 gene point mutations in fluconazole-resistant Candida albicans isolates with rolling circle amplification / 中华皮肤科杂志

Objective To detect point mutations of TAC1 gene in fluconazole-resistant Candida albi-cans isolates with rolling circle amplification (RCA), develop an accurate, rapid and specific assay to detect single nucleotide polymorphisms (SNPs), and to estimate the relationship between mutations of TAC1 gene and resistance to fluconazole. Methods A total of 33 fluconazole-resistant Candida albicana isolates, including 8 strains from America and 25 from Australia, were collected. Four TAC1-specific padlock probes were designed according to previously reported mutations. DNA was extracted from these tested strains, subjected to amplification of three targeted fragments of TAC1 gene with PCR. Then, RCA was performed to detect point mutations of TAC1 gene in resistant Candida albicans strains. At the same time, the target fragments underwent sequencing analysis, and the results of RCA were compared with those of sequencing. Results Two types of resistance-associated mutations were found in 5 out of the 33 fluconazole-resistant strains. Among the 5 strains, 4 were from America, 1 harbored T225A mutation and 4 carried A736V mutation. No related mutation was found in TAC1 gene of 4 fluconazole-sensitive isolates. Conclusions RCA assay could accurately and rapidly detect point mutations of genes. Further studies are required to clarify the relationship between TAC1 point mutations and fluconazole resistance.

The study on the rolling circle amplification for detecting hepatitis B virus covalently closed circular DNA / 中华传染病杂志

Objective To set up the rolling circle amplification (RCA) system for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to evaluate the specificity and sensitivity of this system. Methods Plasmids containing full-length of wild-type HBV genome were treated with restriction enzyme and T4 DNA ligase, and then were concentrated. The DNA fragments were recovered by the nucleic acid purification kit and severed as standard HBV cccDNA. Total DNA was extracted from hepatic tissues of seven chronic hepatitis B patients. RCA method was used to amplify genomes from tissue samples. Standard HBV cccDNA, 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of patients with chronic HBV infection were used as controls to determine the specificity of RCA. Ten-fold serial dilutions of standard HBV cccDNA were used for determining the sensitivity. Results The standard HBV cccDNA was successfully constructed and could be detected by RCA method. HBV cccDNA could be amplified from 2 mg hepatic tissue samples at least of HBV infected patients, and could be detected as low as 1 ×102 copy/μL. cccDNA was not detected in 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of chronic HBV infected patients. Conclusion RCA method can be used for rapid and simple detection of HBV cccDNA with high specificity and sensitivity.

Detection of five common cell culture contaminant Molllcutes using rolling circle amplification / 中华微生物学和免疫学杂志

Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods "Universal" primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute.