Gene Amplification Techniques Research Articles

Amplified DNA heterogeneity assessment with Oxford Nanopore sequencing applied to cell free expression templates.

In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest error rates observed in the amplified DNA. As a secondary validation, we analyzed the emission spectra of sfGFP fluorescent proteins synthesized with amplified DNA using cell free expression. Error-prone polymerase chain reactions confirmed the dependency of reporter protein emission spectra peak broadness to DNA error rates. The presented nanopore sequencing methods serve as a roadmap to quantify the accuracy of other gene amplification techniques, as they are discovered, enabling more homogenous cell-free expression of desired proteins.

Feedback on the detection of SARS-CoV-2 by the point-of-care testing

In order to improve the detection and rapid diagnosis of the SARS-CoV-2 coronavirus, we evaluated the ID NOW™ COVID-19 isothermal gene amplification technique in parallel with the real-time PCR technique (Diasorin) routinely used in the laboratory during a prospective study in the 2020 season. As this technique showed satisfactory sensitivity and specificity of 98% and 97.5% respectively, we then proposed to implement the detection of SARS-CoV-2 coronavirus in the emergency department and maternity as a point-of-care test (POCT) for the 2020-2021 season and to evaluate its clinical and organizational impact. This article summarizes the results obtained and highlights the advantages and limitations of this strategy implemented in the emergency department, particularly in terms of time spent in the department, hospitalization rates, anticoagulant treatment and early isolation of patients, as well as the organizational impact on the maternity unit. Based on this experience, we report on the regulatory constraints that apply when setting up a POCT and the steps required to validate the accreditation in accordance with standard NF EN ISO 22870.

Prevalence and mechanisms of aminoglycoside resistance among drug-resistant Pseudomonas aeruginosa clinical isolates in Iran

BackgroundAminoglycosides have been a cornerstone of the treatment of nosocomial infections caused by Pseudomonas aeruginosa for over 80 years. However, escalating emergence of resistance poses a significant challenge. Therefore, this study aimed to investigate the prevailing patterns of aminoglycoside resistance among clinical isolates of P. aeruginosa in Iran; as well as the underlying resistance mechanisms observed in patients referred to Ardabil hospitals.MethodsA total of 200 isolates from five hospitals were evaluated. The resistance profiles of P. aeruginosa isolates to tobramycin, amikacin, and netilmicin were determined using the disk diffusion method. The capacity of aminoglycoside-resistant isolates to form biofilms was assessed through a phenotypic assay, and the results were confirmed using the gene amplification technique. The presence of genes associated with aminoglycoside resistance was detected using polymerase chain reaction (PCR). Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the expression levels of genes encoding the MexXY-OprM efflux pump and PhoPQ two-component system (TCS).ResultsThe prevalence of aminoglycoside-resistant P. aeruginosa isolates was 48%, with 94.7% demonstrating multidrug resistance (MDR). All aminoglycoside-resistant P. aeruginosa strains exhibited biofilm-forming capabilities and harbored all the genes associated with biofilm production. Among the nine genes encoding 16S rRNA methylase and aminoglycoside-modifying enzymes, three genes were detected in these isolates: aac(6’)-Ib (85.4%), ant(2’’)-Ia (18.7%), and aph(3’)-VI (3.1%). Additionally, all aminoglycoside-resistant P. aeruginosa isolates carried mexY and phoP genes, although the expression levels of mexY and phoP were 75% and 87.5%, respectively.ConclusionGiven the considerably high prevalence of aminoglycoside-resistant P. aeruginosa strains, urgent measures are warranted to transition towards the use of novel aminoglycosides and to uphold vigilant surveillance of resistance patterns.

Amplified DNA Heterogeneity Assessment with Oxford Nanopore Sequencing Applied to Cell Free Expression Templates.

In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest error rates observed in the amplified DNA. As a secondary validation, we analyzed the emission spectra of sfGFP fluorescent proteins synthesized with amplified DNA using cell free expression. Error-prone polymerase chain reactions confirmed the dependency of reporter protein emission spectra peak broadness to DNA error rates. The presented nanopore sequencing methods serve as a roadmap to quantify the accuracy of other gene amplification techniques, as they are discovered, enabling more homogenous cell-free expression of desired proteins.

Highly Multiplexed Reverse-Transcription Loop-Mediated Isothermal Amplification and Nanopore Sequencing (LAMPore) for Wastewater-Based Surveillance.

Wastewater-based surveillance (WBS) has gained attention as a strategy to monitor and provide an early warning for disease outbreaks. Here, we applied an isothermal gene amplification technique, reverse-transcription loop-mediated isothermal amplification (RT-LAMP), coupled with nanopore sequencing (LAMPore) as a means to detect SARS-CoV-2. Specifically, we combined barcoding using both an RT-LAMP primer and the nanopore rapid barcoding kit to achieve highly multiplexed detection of SARS-CoV-2 in wastewater. RT-LAMP targeting the SARS-CoV-2 N region was conducted on 96 reactions including wastewater RNA extracts and positive and no-target controls. The resulting amplicons were pooled and subjected to nanopore sequencing, followed by demultiplexing based on barcodes that differentiate the source of each SARS-CoV-2 N amplicon derived from the 96 RT-LAMP products. The criteria developed and applied to establish whether SARS-CoV-2 was detected by the LAMPore assay indicated high consistency with polymerase chain reaction-based detection of the SARS-CoV-2 N gene, with a sensitivity of 89% and a specificity of 83%. We further profiled sequence variations on the SARS-CoV-2 N amplicons, revealing a number of mutations on a sample collected after viral variants had emerged. The results demonstrate the potential of the LAMPore assay to facilitate WBS for SARS-CoV-2 and the emergence of viral variants in wastewater.

Development of Magnetic-Silica Particles and In-house Buffers Kit for SARS-CoV-2 and CDV RNA Extraction

Since the end of 2019, COVID-19 pandemic caused by the novel SARS-CoV-2 has become a serious problem for the world. Accurate and rapid techniques in testing and tracing are needed to control the virus spreading. Molecular diagnostics through gene amplification techniques, especially PCR, still become the gold standard for SARS-CoV-2 detection, which requires the first step of RNA extraction and purification. The limitations of commercial RNA extraction-purification kits during the pandemic caused a big problem in testing and tracing, especially for developing countries. A simple RNA extraction-purification kit based on magnetic-silica (MAGSi) beads and non-guanidine in-house buffers for RNA virus extraction-purification has been developed. Two types of MAGSi beads with different magnetic nanoparticles (MNPs) content were synthesized through a modified Stöber’s method using the sonication technique. The PCR result shows that both the MAGSi beads and the buffer can be used as a kit for RNA extraction-purification, tested for SARS-CoV-2 and Canine Distemper Virus. Further study shows that MAGSi-1 has better RNA extraction ability, and a higher concentration of RNA has been extracted. This is likely because of the smaller particle size distribution (50–1,500 nm distribution) and higher magnetization (20.2 emu/g) of MAGSi-1 compared to MAGSi-2 with 100–1,700 nm size distribution and 14.2 emu/g magnetization.

Loop mediated isothermal amplification (LAMP) of DNA: An advanced gene amplification technique

The amplification of DNA provides method for gene based specific detection. Besides Polymerase Chain Reaction (PCR), amplification can be achieved by several isothermal techniques. The PCR suffers from various drawbacks like requirement of laboratory and expertise to perform. Loop Mediated Isothermal Amplification (LAMP) of DNA is one of the promising isothermal techniques rapidly gaining popularity among researchers. The technique requires Bst DNA polymerase and a set of four primers recognizing 6-8 regions of target DNA. LAMP technique of DNA gained surprising popularity for amplifying a desired gene at constant temperature between 60-65 °C & maintain a constant temperature can be easily fulfilled by a water bath or a heat block. The LAMP amplification of the desired DNA fragment can be achieved as high as 109 copies in merely 30 min with higher specificity as compared with the existing PCR technique. The technique of LAMP averts the use of costly thermal cycler instrument, facilitating establishment of a field-friendly technique and is promising technique for gene detection in a resource-limited laboratory.

Efficacy and Safety of rCCK96-104PE38 Targeted Drug in the General Surgical Treatment of Colon Cancer.

To evaluate the clinical efficacy and safety of the rCCK96-104PE38 targeted drug in patients with colon cancer in general surgery, data of 80 patients with colon cancer who were admitted to the hospital from April 2019 to July 2021 were selected and randomly divided into the treatment group and the control group, with 40 cases in each group. Patients in the treatment group were treated with the rCCK96-104PE38 targeted drug, and those in the control group were treated with oxaliplatin. The treatment efficiency and incidence of adverse reactions were compared between the two groups. The inverse cholecystokinin (CCK96-104) was fused with pseudomonas aeruginosa exotoxin (PE38 toxin) through the gene amplification technique to construct a prokaryotic expression vector. Then, the rCCK96-104PE38 was purified by Ni-nitrilotriacetate (Ni-NTA) affinity chromatography, and the antitumor activity of rCCK96-104PE38 was verified. The results showed that the amplified rCCK96-104PE38 sequence was correct and the pET-28a prokaryotic expression system was adopted to successfully achieve active expression. The purified recombinant protein could induce the apoptosis of colon cancer cells in vitro and inhibit tumor growth in vivo. The total effective rate in the treatment group (80%, 32/40) was higher than that in the control group (60%, 24/40) (P < 0.05). To sum up, the recombinant toxin rCCK96-104PE38 could not only specifically adsorb the colon cancer cells with high expression of CCK2R but also effectively inhibit tumor tissue growth and proliferation. Besides, the rCCK96-104PE38 protein had a good anticancer effect that helped effectively reduce the incidence of adverse reactions in patients, which was worthy of promoting.

Mitochondrial Cox-1 Barcoding For The Identification and Phylogenetic Analysis of Sarcophaga (Liosarcophaga) dux Recorded From Jeddah City of Saudi Arabia

This study was aimed to identify and phylogenetically analyze the sarcophagid flies from Jeddah (Saudi Arabia) on the basis of mitochondrial cytochrome-C-oxidase subunit-1 (COX-1) gene. Five flesh fly samples were collected from different locations of Jeddah region. All the samples were first morphologically identified as Sarcophaga dux and confirmed on the basis of COX-1 gene amplification and sequencing techniques using universal primers LCO1490 and HCO2198. For phylogenetic analysis, 5 sequences were checked by COX-1 service on barcode of life data system (BOLD) database. To determine the phylogenetic relationships and genetic variability between 5 collected and BOLD samples, large Neighbor-joining tree comprising of the collected samples and all the available S. dux BOLD samples were constructed. The 32 most related BOLD sequences were used to construct maximum likelihood (ML) and Neighbor-joining trees. The phylogenetic analysis of COX-1 gene sequences revealed significant match with S. dux sequences from BOLD database: GQ254446 (Australia), MG780139 (India), AY879255 (China), JN869983 (Malaysia) and KC249664 (Egypt). This is the first report of the flesh fly species recorded in the Western regions of Saudi Arabia. Results indicated that the genetic diversity and phylogenetic relationship analysis of the collected S. dux samples have an important role in understanding the evolutionary processes of Sarcophaga cryptic species in Western regions of Saudi Arabia.

An Improved Method for the Extraction of Nucleic Acids from Plant Tissue without Grinding to Detect Plant Viruses and Viroids.

Gene amplification techniques such as polymerase chain reaction (PCR) are widely used for the diagnosis of plant diseases caused by viruses and viroids. It is preferable that sample preparation methods for PCR or reverse transcription (RT) PCR are rapid, straightforward, and inexpensive. We previously reported a method for the extraction of nucleic acids without mechanical tissue grinding using a buffer containing potassium ethyl xanthogenate (PEX) to detect viroid RNAs. In the present report, the previous PEX method was improved and simplified. In the simplified PEX (SPEX) method, the process of PEX buffer treatment for plant cell wall disruption is improved to one step of incubation at 80 °C for 10 min, instead of three steps that took more than 26 min at 65 °C in the previous method. Total nucleic acids could be extracted from fresh, frozen, or dried leaves of a cultivar or wild species of tobacco, tomato, citron, hop plants, and pericarps of persimmon fruits by the SPEX method. Several RNA viruses and viroids were successfully detected from the extracted nucleic acids together with an internal mRNA by RT-PCR. The SPEX method may be useful for detecting not only viruses and viroids, but also other plant pathogens.

Retrospective diagnosis of lymphatic tuberculosis in frozen samples using two genetic amplification methods, Xpert® MTB/RIF ULTRA and Abbott RealTime MTB Assay.

ObjectivesThe main objective of the present study is to assess the sensitivity and specificity of a retrospective diagnostic of lymphatic tuberculosis (LTB), testing frozen samples using gene amplification PCR methods. The secondary objective was to compare the results of two different commercial tuberculosis gene amplification methods for this purpose.Material and methodsWe retrospectively studied 38 frozen samples, previously processed for mycobacterial culture between January 2014 and August 2019. The results of the previous cultures were: 21 samples positive for Mycobacterium tuberculosis complex (MTB) (5 being smear positive), 7 samples culture positive for Mycobacterium avium-intracellulare complex and 10 samples which were mycobacterial culture negative and discarded for LTB diagnosis, used as controls. The samples were processed using two gene amplification methods: Xpert® MTB/RIF Ultra (Cepheid) and Abbott RealTime MTB Assay (Abbott).ResultsCompared to initial culture results the sensitivity and specificity of Xpert® MTB/RIF Ultra were 57.1% and 100% and 52.3 % and 92.5%, respectively for the Abbott RealTime MTB assay. The differences were not statistically significant. In addition, there were no differences according to the period of freezing.ConclusionsGene amplification of frozen samples confirmed the diagnosis of lymphatic TB in almost 60% of cases, allowing retrospective diagnosis in initially non suspected cases. Both gene amplification techniques tested were equally useful.

DEPIS: A combined dielectrophoresis and impedance spectroscopy platform for rapid cell viability and antimicrobial susceptibility analysis

Antimicrobial resistance (AMR) is caused by inappropriate or excessive antibiotic consumption. Early diagnosis of bacterial infections can greatly curb empirical treatment and thus AMR. Current diagnostic procedures are time-consuming as they rely on gene amplification and cell culture techniques that are inherently limited by the doubling rate of the involved species. Further, biochemical methods for species identification and antibiotic susceptibility testing for drug/dose effectiveness take several days and are non-scalable. We report a real-time, label-free approach called DEPIS that combines dielectrophoresis (DEP) for bacterial enrichment and impedance spectroscopy (IS) for cell viability analysis under 60 min. Target bacteria are captured on interdigitated electrodes using DEP (30 min) and their antibiotic-induced stress response is measured using IS (another 30 min). This principle is used to generate minimum bactericidal concentration (MBC) plots by measuring impedance change due to ionic release by dying bacteria in a low conductivity buffer. The results are rapid since they rely on cell death rather than cell growth which is an intrinsically slower process. The results are also highly specific and work across all bactericidal antibiotics studied, irrespective of their cellular target or drug action mechanism. More importantly, preliminary results with clinical isolates show that methicillin-susceptible Staphylococcus aureus (MSSA) can easily be differentiated from methicillin-resistant S. aureus (MRSA) under 1 h. This rapid cell analyses approach can aid in faster diagnosis of bacterial infections and benefit the clinical decision-making process for antibiotic treatment, addressing the critical issue of AMR.

Transient reprogramming of crop plants for agronomic performance.

The development of a new crop variety is a time-consuming and costly process due to the reliance of plant breeding on gene shuffling to introduce desired genes into elite germplasm, followed by backcrossing. Here, we propose alternative technology that transiently targets various regulatory circuits within a plant, leading to operator-specified alterations of agronomic traits, such as time of flowering, vernalization requirement, plant height or drought tolerance. We redesigned techniques of gene delivery, amplification and expression around RNA viral transfection methods that can be implemented on an industrial scale and with many crop plants. The process does not involve genetic modification of the plant genome and is thus limited to a single plant generation, is broadly applicable, fast, tunable and versatile, and can be used throughout much of the crop cultivation cycle. The RNA-based reprogramming may be especially useful in plant pathogen pandemics but also for commercial seed production and for rapid adaptation of orphan crops.

Development of magnetic bead based sample extraction coupled polymerase spiral reaction for rapid on-site detection of Chikungunya virus

The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology. However the major limitation in implementation of available molecular detection assays is the non availability of field deployable nucleic acid isolation platform coupled with gene amplification technique. The rapid and early molecular detection is crucial for employing effective measure against many viral infections. The re-emergence of chikungunya virus (CHIKV) has led to epidemics since 2004 in several parts of the world including India. The main association of CHIKV with severe arthritis and long-lasting arthralgia and closely mimics symptoms of Dengue and Zika virus infection requiring laboratory confirmation. In this study, a simple magnetic bead based ribonucleic acid extraction method was optimized, which was coupled with isothermal polymerase spiral reaction (PSR) technique for early and rapid detection. Subsequently, the polymerase spiral reaction reagents were converted to dry down format that led to a rapid user friendly field compatible sample processing to answer method for rapid and onsite detection of Chikungunya virus. Both the methods were evaluated with a panel of clinical samples. The sensitivity of the assays were compared with available commercial viral RNA extraction platform and qRT-PCR. The in-house nucleic acid extraction system based on magnetic bead followed by dry down RT-Polymerase Spiral Reaction assay was found to be highly sensitive with 10 copies of RNA as limit of detection in CHIKV clinical specimens. With respect to other closely related viruses no cross reactivity was observed. This novel methodology has the potential to revolutionize the diagnosis of infectious agents in resource limited settings around the world.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) pathway dominates nitrate reduction processes in rhizosphere and non-rhizosphere of four fertilized farmland soil

Nitrate (NO3−) reduction partitioning between denitrification, anaerobic ammonium oxidation (anammox), denitrifying anaerobic methane oxidation (DAMO), and dissimilatory nitrate reduction to ammonium (DNRA), can influence the nitrogen (N) use efficiency and crop production in arid farmland. The microbial structure, function and potential rates of denitrification, anammox, DAMO and DNRA, and their respective contributions to total NO3− reduction were investigated in rhizosphere and non-rhizosphere soil of four typical crops in north China by functional gene amplification, high-throughput sequencing, network analysis and isotopic tracing technique. The measured denitrification and DNRA rate varied from 0.0294 to 20.769 nmol N g−1 h−1and 2.4125–58.682 nmol N g−1 h−1, respectively, based on which DNRA pathway contributed to 84.44 ± 14.40% of dissimilatory NO3− reduction, hence dominated NO3− reduction processes compared to denitrification. Anammox and DAMO were not detected. High-throughput sequencing analysis on DNRA nrfA gene, and denitrification nirS and nirK genes demonstrated that these two processes did not correlate to corresponding gene abundance or dominant genus. RDA and Pearson's correlation analysis illustrated that DNRA rate was significantly correlated with the abundance of Chthiniobacter, as well as total organic matter (TOM); denitrification rate was significantly correlated with the abundance of Lautropia, so did TOM. Network analysis showed that the genus performed DNRA was the key connector in the microbial community of dissimilatory nitrate reducers. This study simultaneously investigated the dissimilatory nitrate reduction processes in rhizosphere and non-rhizosphere soils in arid farmland, highlighting that DNRA dominated NO3− reduction processes against denitrification. As denitrification results in N loss, whereas DNRA contributes to N retention, the relative contributions of DNRA versus denitrification activities should be considered appropriately when assessing N transformation processes and N fertilizer management in arid farmland fields.

CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage.

Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported. The core of CUT-LAMP is the engineering of the forward or backward inner primer in the target-independent region, which makes the LAMP products contain a protospacer adjacent motif (PAM) site for the CRISPR/Cas9 recognition. For the CUT-LAMP reaction, cross-contamination can be efficiently cleaved by the corresponding Cas9/sgRNA, but the target gene can get rid of digestion due to the lack of a PAM site near the recognition region. CUT-LAMP shows impressive contamination resistance but does not significantly increase procedure complexity; thus, it represents a simple and versatile toolkit facilitating the adoption by open- and closed-tube detection format.

Expression and clinical significance of galectin-9 in different cervical lesions and different types of human papillomavirus infection

Objective To analyze the expression differences and clinical significance of galectin 9(Gal-9) in different cervical lesions and human papillomavirus(HPV) type infection. Methods From May 2016 to April 2018, 42 cases of cervical cancer(CC group), 45 cases of high level cervical intraepithelial neoplasia (HSIL group), 45 cases of lower cervical intraepithelial neoplasia (LSIL group) in Yangzhou Maternal and Child Health-Care Hospital were selected, and 45 cases of normal outpatient medical examination in our hospital were selected as the control group(NC group). Enzyme-linked immunosorbent assay(ELISA) method was used to detect the serum level of Gal-9.The gene amplification technique and the principle of diversion hybridization were used to detect the 37 types of HPV. Results Of women with cervical lesions, 50 cases(28.3%) were 50 years or more, the CC group had 32 cases (76.2%), HSIL group had 10 cases (22.2%), LSIL group had 8 cases (17.8%), the age of CC group compared with HSIL group and LSIL group had significant differences(t=7.959, 10.440, all P<0.01). The serum level of Gal-9 in CC group [(22.27±1.72)ng/L], HSIL group [(15.43±2.14)ng/L], LSIL group [(15.24±1.89)ng/L] was significantly higher than that in NC group [(12.08±2.13)ng/L](t=16.360, 18.100, 24.440, all P<0.05). The HPV infection rate in patients with cervical lesions reached 84.84%(112/132), the positive rate of HPV16 in CC group was 83.3%(35/42), HSIL group was dominated by HPV16(28.8%), HPV33(20.0%), HPV52(17.7%) and HPV58(13.3%), LSIL group was dominated by HPV52(26.7%), HPV58(24.4%), HPV16(20.0%) and HPV18(8.9%). There was no statistically significant difference in the levels of Gal-9 of HPV16, HPV58, HPV52 and HPV33 patients, indicated that there was no statistically significant correlation between Gal-9 level and the type of HPV infection. Conclusion Early intervention in patients with high HPV infection and Gal-9 levels can help reduce the incidence of cervical cancer. Key words: Galectins; Uterine cervical diseases; Uterine cervical neoplasms; Carcinoma, squamous cell; Cervical intraepithelial neoplasia; Human papillomavirus; Genotype; Gene expression

Utilizing multiplex fluor LAMPs to illuminate multiple gene expressions in situ.

In situ gene expression detection is the best way to determine temporal and spatial differences in gene expression. However, in situ hybridization procedures are inherently difficult to execute and typically suffer from degradation of sample tissues, limited sensitivity to genes with low expression, high background, and limitation to single gene detections. We propose to utilize an isothermal gene amplification technique, LAMP (Loop-Mediated Isothermal Amplification), to solve these problems in a novel way. LAMP greatly amplifies the signal of expressed genes and can use multiple sets of primers and different fluorescent-labeled probes to produce multiplex gene detection. LAMP is a rapid, isothermal reaction that reduces the handling and degradation of tissue by cutting down on the washing steps required by other methods. Using this technique, we have successfully amplified 3 target genes, have produced positive fluorescent in situ results simultaneously for two genes. We have also demonstrated that LAMP can be used to exploit standard NBT/BCIP (nitro-blue tetrazolium chloride/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) detection of single expression. In situ LAMP is a robust and applicable method that can be exploited for detection of gene expression in plant species, as well as in animals and bacteria.

Development of probe-based real-time loop-mediated isothermal amplification for detection of Brucella.

To develop a rapid, sensitive and specific diagnostic assay for the detection of Brucella. The probe-based RT-LAMP was carried out by using a set of four or six primers and different LAMP chemicals to compare its results with real-time PCR. Detection of gene amplification is done within 40min and can be seen by amplification curve, turbidity and addition of DNA-binding dye at the end of the reaction results in colour difference under normal day light and in UV. The sensitivity of probe-based real-time LAMP assay was found 10-fold higher than Taqman-based qPCR. The specificity of the developed assay was validated by the absence of any cross-reaction with other pathogenic bacteria. The developed probe-based RT-LAMP assay is extremely rapid, cost effective, highly specific and sensitive, and has potential usefulness for rapid Brucella surveillance. The developed probe-based RT-LAMP is a powerful gene amplification technique which is a specific, fast diagnostic tool for early detection and identification of Brucella.

Viral Hepatitis B: Seroprevalence and Genetic Diversity in Blood Donors North of Congo

Introduction: Viral hepatitis B is the most formidable and confusing of all viral hepatitis, given its cirrhogenic and carcinogenic potential. The objective of the study is to characterize the molecular profile of hepatitis virus B in northern Congo. Materials and Methods: This was a descriptive and cross-sectional study that was carried out between January and September 2014, that is 9 months, in the establishments of the National Center for Blood Transfusion (CNTS) of the 4 departments of the north of the Congo. Epidemiological, serological and molecular variables (HBsAg, HBV DNA, genotypes and subtypes) were studied. The HBs antigen was searched by rapid test and confirmed by ELISA. For all positive donors, conventional and specific gene extraction and amplification techniques were performed for the identification of genotypes and subtypes from the serum. Results: A total of 892 donors were included. The average age was 35.36 ± 12.36 years with extremes ranging from 18 to 65 years old. The sex ratio (M/F) was 3.3. The prevalence of HBV was 8.6%. The viral DNA of HBV was amplified in 83.1%. Four genotypes were found: E (40.6%), A (3.1%), B (3.1%) and C (1.6%). Two cases of co-infection were identified: E/A (15.6%) and B/C (1.6%). Subgenotypes A1, B2 and C1 were highlighted. Conclusion: The prevalence of HBV is high, HBV infection remains a major public health problem for blood donors in Congo. Genotypes E, A, B, C as well as subtypes A1, B2, C1 have been identified in northern Congo.