Conventional Gel Electrophoresis Research Articles

Customizable BAC-based DNA markers for pulsed-field gel electrophoresis

DNA markers are used as a size reference and sample loading control during gel electrophoresis. Most markers are designed for conventional gel electrophoresis to separate DNA smaller than 20 kb. For larger molecules, pulsed-field gel electrophoresis (PFGE) marker is required. Limited PFGE markers are available because large DNA are prone to nicking and degradation, causing smeary bands. Here, we developed a robust marker based on bacterial artificial chromosomes (BACs) with bands up to 184 kb. This marker could consistently confer intense and distinct bands for accurate gel analysis in molecular biology studies, laboratory validations or clinical diagnosis.

Isolation and assessment of Pseudomonas aeruginosa and Bacillus cereus as PCB-degrading bacteria in environmental bioremediation

Classical methods of treatment of polychlorinated biphenyls (PCBs) in environmental matrices such as soil encapsulation, high-temperature heating, thermal desorption, and chemical decontamination, have been costly, and they leave behind problems of environmental footprints. They must therefore give way to bioremediation techniques which are less costly and result in eco-friendly products. The aim of the study was to assess the suitability of PCB degrading bacteria (using published data) from their environmental matrix for use in environmental bioremediation. Initial isolation of the bacteria was obtained by culturing ash samples taken from the combustion chamber of an MP400 Incinerator of infectious waste at Komfo Anokye Teaching hospital (KATH) in Kumasi, Ghana, and composite soil samples taken from a transformer-servicing site at an electricity substation in Sunyani, Ghana. Test for thermotolerance was carried out by the culturing of bacteria from the soil samples and exposing them to higher temperatures (55 °C and 65 °C) to assess their resilience in an incubator for 48 h. Growing cultures were sub-cultured, after which a cocktail of bacteria isolate characterization assays was carried out on the growing isolates. These included biochemical tests on the bacterial suspensions using the analytical profile index (API 20E) for examining Enterobacteriaceae and other non-fastidious bacteria. This was followed by MALDI-ToF-MS analyses of the bacterial strains to profile and image their proteins. A molecular step was needed which was the conventional polymerase chain reaction (PCR) method, using specific primer pairs, to amplify and accentuate sections of the bacterial DNA extracts. Results of the study showed a confirmed sample site in Ghana for harvesting bacteria capable of degrading polychlorinated biphenyls (PCBs). MALDI-ToF-MS analyses revealed the possible species as Pseudomonas aeruginosa and Bacillus cereus. The conventional PCR and gel electrophoresis steps confirmed the presence of bph genes encoding the 2,3-dioxygenase enzymes in bacteria with PCB-degrading ability as documented in published data. The serial dilution of the bacterial suspensions was modified from 10−7 to 10−5 which was enough for distinct colonies to be counted, and it was a novel contribution to environmental microbiology. The confirmation of Pseudomonas and Bacillus species isolated from a transformer oil-impacted soil in this study shows promise in growing these strains in various environmental media such as in a gaseous medium to mineralize gas-phase PCBs from combustion smoke or in bottom ash residues to degrade solid-phase PCBs. It can also be applied in bioremediation of waste liquid systems containing PCBs.

Frequency of OXA-Type Carbapenemases among Carbapenem-Resistant Acinetobacter baumannii in Clinical Isolates from Adult Intensive Care Unit in India

Abstract Purpose Acinetobacter baumannii is a highly virulent bacteria in modern health care, with a high ability to acquire antimicrobial resistance. Carbapenemases production appears to be the most common mechanism involved in drug resistance to carbapenem. As the prevalence of carbapenem-resistant A. baumannii was high in intensive care unit (ICU) patients, this study was designed to find the frequency of oxacillinases (OXA) genes including OXA-23, OXA-24, OXA-51, and OXA-58. Materials and Methods A clinical specimen was collected from patients admitted to the adult ICU. DNA was isolated from carbapenem-resistant A. baumannii and amplified using conventional polymerase chain reaction technique and gel electrophoresis for visualization of results. Results The frequency of the OXA-23 gene was high with 87.5%, followed by OXA-51 gene with 73.2%. All 56 isolates were negative for the OXA-24 and OXA-58 genes. We also found that both OXA-23 and OXA-51 genes coexisted in 40 (71.4%) isolates. No significant difference was found between drug-resistant genes (OXA-23 and OXA-51) and clinical outcomes. The relationship between the presence of OXA gene was compared between survivors and nonsurvivors, which was found out to be nonsignificant. The presence of OXA genes showed no significant increase in the length of hospital stay. The significant association between acute physiology and chronic health evaluation IV scores and clinical outcome was calculated, and it was evident in the comparison of the discharged and died groups. Conclusion Early detection of these drug-resistant genes by molecular methods is essential in decreasing the spread of carbapenem-resistant A. baumannii.

A label-free electrochemical biosensor for the detection of alpha-thalassemia 1 (SEA deletion) carriers using screen-printed carbon electrodes

A label-free electrochemical DNA biosensor based on electrochemical impedance spectroscopy (EIS) biosensor has been extensively developed for diagnosing human genetic diseases. However, its application has been limited to simulated target DNA. The aim of this study was to develop an EIS biosensor that can identify carriers of alpha (α)-thalassemia 1 with Southeast Asia (SEA) deletion. The biosensor was coupled with screen-printed carbon electrodes (SPCEs) and Hoechst 33,258 dye. To determine the optimal conditions and cutoff criteria, we evaluated forty samples with known DNA genotypes. Our findings suggested that the cutoff value for identifying α-thalassemia 1 (SEA deletion) carriers was more than 26.84 kΩ of negative imaginary impedance (-Z″) and 48.83 kΩ of charge transfer resistance (Rct). The sensitivity of the developed EIS biosensor was comparable to that of conventional gel electrophoresis, with no cross-reactivity observed in α-thalassemia 1 (THAI deletion), α-thalassemia 2 (3.7 deletion), α-thalassemia 2 (4.2 deletion), and beta (β)-thalassemia carriers. The diagnostic potential of the developed EIS biosensor was evaluated using 81 clinical blood samples, demonstrating 100% sensitivity, 88.2% specificity, 83.3% positive predictive value (PPV), 100% negative predictive value (NPV) and 92.6% accuracy for Rct measurement. The developed EIS biosensor presents an attractive screening method for the detecting α-thalassemia 1 (SEA deletion) carriers due to its simplicity, cost-effectiveness, and 45-fold reduction in turnaround time compared to conventional gel electrophoresis. Consequently, the identification of α-thalassemia 1 (SEA deletion) carrier through this approach represents the most effective strategy for the management and prevention of the most severe form of thalassemia.

Occurrence of mcr genes and alterations in mgrB gene in intrinsic colistin- resistant Enterobacterales isolated from chicken meat samples

BackgroundTolerance of gram negative pathogens toward last resort colistin is mediated by mcr genes and alterations in chromosomal mgrB via modification of lipopolysaccharide through the PmrAB and PhoPQ component system. Proteus sp., Morganella sp., Neisseria sp., Burkholderia sp. and Providencia sp. are intrinsic resistant to colistin drug. Recent reports have shown that colistin intrinsic resistant organisms harbor and act as reservoirs for mcr genes. AimTo evaluate the presence of mcr-1 to mcr-5 genes and alterations in mgrB gene in intrinsic colistin-resistant gram negative bacteria isolated from chicken meat samples in Coimbatore district, Tamil Nadu, India. MethodsOne hundred chicken meat samples were collected during 2019–20. Samples were enriched and plated on MacConkey agar supplemented with colistin (2 μg/ml). The bacterial isolates were then identified using biochemical tests. DNA were extracted from isolates using the thermal lysis method. mcr-1 to mcr-5 and mgrB genes was detected using conventional PCR and agarose gel electrophoresis methods. Result and conclusionThe presence of mcr-1 to mcr-5 genes was found to be 23 % (23/100). mcr-1 and mcr-5 genes were not detected in sample isolates. 17/23 samples positive for mcr genes were also found to be carrying alterations in mgrB gene. Phenotypic characterization of these isolates revealed that these bacteria were belonging to colistin intrinsic resistant gram negative bacteria such as Proteus sp., Providencia sp., and Morganella sp. Intrinsic resistant bacteria could act as a potential reservoir and disseminate mcr genes to the colistin sensitive non-intrinsic pathogens of clinical importance in the environment.

Biosensor for PCR amplicons by combining split trehalase and DNA-binding proteins: A proof of concept study

The use of polymerase chain reaction (PCR) technology in low-cost settings has gained significant attention due to its ability to amplify and detect specific bacterial pathogen genes, aiding in the diagnosis of infectious diseases. PCR amplicons can be visualized by conventional endpoint agarose gel electrophoresis and fluorochrome-enabled real-time PCR. However, this is not practical in on-field tests due to cumbersome instrumentation, labor-intensive reaction preparation, and long time-to-results. Many studies have combined microfluidic devices or electrochemical dyes with PCR technology to enhance in-field operability. However, the high cost of manufacturing high-precision microfluidic chips and the dependence on non-portable readout equipment limit their further development. In this paper, we present a proof-of-principle study of a novel method combining split enzyme technology and DNA-binding proteins for the convenient and efficient detection of amplified genetic material from bacterial pathogens. The amplicon binding split trehalase assay (ABSTA) relies on incorporating specific recognition sequences of DNA-binding protein SpoIIID in tandem within one of the PCR primers. Applied by a Gram-type specific PCR assay, ABSTA was capable of discriminating Staphylococcus devriesei and Escherichia coli in <90 min after colony PCR amplicons bound split trehalase fragments-fused SpoIIID and triggered split enzyme complementation. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. The glucose produced by restored enzymatic activity was detectable by glucometer. With limited requirements for reaction preparation and the compatibility of ABSTA with commercially available handheld glucometers, this test platform has substantial potential to be implemented into a future point-of-care (POC) diagnostic tool for detecting pathogen specific genes with further improvement.

Simultaneous detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacteria coexisting in patients with pulmonary tuberculosis by single-tube multiplex PCR.

In clinical diagnostics, molecular methods are used to detect Mycobacterium tuberculosis bacilli (MTB) and to distinguish them from non-tuberculous mycobacteria (NTM). They are also used to make the right treatment decision for the patient as soon as possible. The aim of this study was to establish a rapid and novel multiplex PCR (mPCR) assay for the detection and differentiation of MTB and NTM in a single tube. 100 sputum samples positive for acid-fast bacilli (AFB) were included in this study. Mycobacterial culture, biochemical tests, and antibiotic susceptibility testing were performed on samples. After alkaline decontamination, total DNA was extracted from the samples. A primer pair targeting the rpoB gene, encoding the beta-subunit of RNA polymerase, was used to detect MTB and NTM, amplifying a 235-bp fragment of MTB and a 136-bp sequence of NTM. A pair of primers targeting a 190-bp fragment of the IS6110 region of MTB was also used to confirm the results. The sensitivity and specificity of the mPCR assay were evaluated using DNA extracted from standard strains. The amplified products were then analyzed by conventional agarose gel electrophoresis. Of 100 AFB smear-positive sputum samples, 92 MTB DNA, 7 NTM DNA, and one mixed-infection sample were identified in a single tube using mPCR assay. There was no correlation between the AFB degree of smear positivity and PCR results. Of seven NTM isolates, 6 (86%) were resistant to rifampin, isoniazid, and ethambutol, the three first-line anti-tuberculosis drugs. A single-tube mPCR assay based on the rpoB gene provides a rapid and reliable means of detecting and differentiating MTB and NTM in sputum specimens.

Substantial Slowing of Electrophoretic Translocation of DNA through a Nanopore Using Coherent Multiple Entropic Traps.

One of the major challenges in the technology of sequencing DNA using single-molecule electrophoresis through a nanopore is to control the translocation of the macromolecule across the pore in order to allow sufficient time for accurate sequence reading at limited recording bandwidths. If the translocation speed is too fast, the signatures of the bases passing through the sensing region of the nanopore overlap in time, presenting difficulties in accurately identifying the bases in a sequential manner. Even though several strategies, such as enzyme ratcheting, have been implemented to reduce the translocation speed, the challenge to achieve a substantial reduction in the translocation speed continues to be of paramount significance. Toward achieving this goal, we have fabricated a nonenzymatic hybrid device that can reduce the translocation speed of long DNAs by more than 2 orders of magnitude, in comparison with the current status of the art. This device is made of a tetra-PEG hydrogel that is chemically anchored to the donor side of a solid-state nanopore. The idea behind this device is based on the recent discovery of the topologically frustrated dynamical state of confined polymers, whereby the front hydrogel matter of the hybrid device provides multiple entropic traps for a single DNA molecule holding it back against the electrophoretic driving force that pulls the DNA through the solid-state nanopore portion of the device. As a demonstration of slowing DNA translocation by a factor of about 500, we find the average translocation time realized in the present hybrid device for 3 kbp DNA as 23.4 ms, whereas the corresponding time for the bare solid-state nanopore under otherwise identical conditions is 0.047 ms. Our measurements on 1 kbp DNA and λ-DNA show that such a slowing down of DNA translocation with our hybrid device is general. An additional feature of our hybrid device is its incorporation of all features of the conventional gel electrophoresis to separate different DNA sizes in a clump of DNAs and to streamline them in an orderly and slow manner into the nanopore. Our results suggest the high potential of our hydrogel-nanopore hybrid device in further advancing the single-molecule electrophoresis technology to accurately sequence very large biological polymers.

A Collection of RPA-Based Photoelectrochemical Assays for the Portable Detection of Multiple Pathogens.

Portable, ultrasensitive, and simultaneously quantitative detection of the nucleic acids of multiple foodborne pathogens is critical to public health. However, the current testing methods depend on costly equipment and tedious amplification steps. In this study, we propose a photoelectrochemical (PEC) biosensor combined with recombinase polymerase amplification (RPA) technology (RPA-PEC) for the rapid detection of multiple foodborne pathogens under irradiation of 980 nm light. In particular, two working surfaces were designed on homemade three-dimensional screen-printed paper-based electrodes. The genomic DNAs of Escherichia coli O157:H7 and Staphylococcus aureus was initiated by RPA on the corresponding electrode surfaces, thus forming a lab-on-paper platform. Using the formed DNA-PEC signaler, photocurrents were achieved at 37 °C after only 20 min of RPA. The detection performance was superior to that of conventional agarose gel electrophoresis, with detection limits of 3.0 and 7.0 copies/μL for E. coli O157:H7 and S. aureus, respectively. Our study pioneers a new RPA-PEC method for foodborne pathogens and provides directions for the construction of lab-on-paper platforms for the portable detection of multiple nucleic acids.

Detection of Babesia bovis using loop-mediated isothermal amplification (LAMP) with improved thermostability, sensitivity and alternative visualization methods

Bovine babesiosis is one of the most economically important tick-borne diseases in tropical and subtropical countries. A conventional microscopic diagnosis is typically used because it is inexpensive and expeditious. However, it is highly dependent on well-trained microscopists and tends to be incapable of detecting subpatent and chronic infections. Here, we developed a novel nucleic acid-based amplification method using loop-mediated isothermal amplification (LAMP) in conjunction with a colori-fluorometric dual indicator for the rapid and accurate detection of Babesia bovis based on the mitochondrial cytochrome b gene. We aimed to improve the thermostability, sensitivity, specificity, and alternative visualization of LAMP-based methods. We assessed its diagnostic performance compared to two conventional PCR agarose gel electrophoresis (PCR-AGE) methods. The thermostability of LAMP reaction mixtures and DNA templates in variable conditions was also assessed. In addition, we evaluated alternative visualization methods using different light sources including neon, LED, and UV lights. We found that the LAMP-neon was ten times more sensitive than the PCR-AGE, while the LAMP-LED and LAMP-UV were 1,000 times more sensitive. The current LAMP method showed no cross-amplification with uninfected cattle DNA or other common blood parasites in cattle, including Babesia bigemina, Theileria orientalis, Anaplasma marginale, and Trypanosoma evansi. In addition, the developed LAMP method has good thermostability and the potential for on-site utility as B. bovis DNA could still be detected up to 72 h after initial preparation. Our findings suggested that the developed LAMP method provides an alternative approach for B. bovis detection with sensitivity higher than PCR-AGE diagnostics, high specificity, and the flexibility to use neon, LED, and UV light sources for positive signal observations.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

2D-DIGE Proteomic Analysis of Mouse Liver Within 1 Week.

Several years have passed since LC (liquid chromatography)-MS (mass spectrometry) became the mainstream for proteomic analysis; however, conventional fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) continues to be an important technology that enables rapid and direct visualization of hundreds to thousands of proteins and their quantitative analyses. We can get global proteomic views using 2D-DIGE within 3 days and then identify proteins with differential expression levels using MALDI-TOF/MS and MASCOT search engine. Here, we describe our routine 2D-DIGE proteomic analysis of the liver isolated from mice in pathological conditions within 1 week.

Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method.

Simple SummaryPorcine epidemic diarrhea (PED) is a severe disease which has led to tremendous economic losses in the swine industry all over the world. The early detection of its pathogen (PEDV) is vital to prevent and cure this disease. Here, we report the development of a new visual diagnostic test for PEDV—the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. This new assay proved to be specific and sensitive when applied to clinical specimens.Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 °C and 3 min at 80 °C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/μL. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis.

Conventional Gel Electrophoresis-Resolvable Insertion/Deletion Markers for Individual Identification and Analysis of Population Genetics in Red-Crowned Cranes in Eastern Hokkaido, Japan.

Simple SummaryThe red-crowned crane is an endangered bird species in the Far East Eurasian continent and Hokkaido, Japan. Individual identification has been achieved with banding and installed transmitters in only a few cranes, though individual identification is essential for various assessments of cranes on their behavioral characteristics, for example. An insertion/deletion (InDel) mutation is a mutation ranging from 1 to 50 bp and is very useful for genetic studies. If primer sets across InDels (>20 bp) can be designed, an InDel polymorphism can be determined with conventional agarose gel electrophoresis, as reported in some plant species. We found a sufficient number of InDel primer sets to be used for individual identification and analysis of population genetics of red-crowned cranes in Hokkaido. The method has additional advantages, such as convenience and low cost, without sequencing and an expensive apparatus.Red-crowned crane Grus japonensis is an endangered species in two separate populations: the mainland population in the Eurasian continent and the island population in eastern Hokkaido, Japan. We found 11 insertion/deletion (InDel) markers in the genome of the red-crowned crane and designed primer sets across these InDels that can be analyzed with conventional agarose gel electrophoresis. Sixty-six samples of whole blood and skeletal muscle obtained from red-crowned cranes, including 12 families in eastern Hokkaido from 1994 to 2021, showed different patterns in gel images of 11 InDel PCR reactions except for two pairs. The combined non-exclusion probability of the 11 markers indicates that individuals can be determined with a probability of 99.9%. In 39 non-relative chicks, the expected heterozygosity (He) was 0.316, suggesting low genetic diversity. This might not be caused by high levels of inbreeding since the average FIS was not significantly different from zero (0.095, p = 0.075). The results suggest that the 11 InDel primer sets can be used for fairly accurate individual identification as well as genetic population analyses in red-crowned cranes in the island population.

Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation.

The physical stability of bacterial chromosomes is important for their in vitro manipulation, while genetic stability is important in vivo. However, extracted naked chromosomes in the open circular form are fragile due to nicks and gaps. Using a nick/gap repair and negative supercoiling reaction (named SCR), we first achieved the negative supercoiling of the whole genomes extracted from Escherichia coli and Vibrio natriegens cells. Supercoiled chromosomes of 0.2–4.6 megabase (Mb) were separated by size using a conventional agarose gel electrophoresis and served as DNA size markers. We also achieved the enzymatic replication of 1–2 Mb chromosomes using the reconstituted E. coli replication-cycle reaction (RCR). Electroporation-ready 1 Mb chromosomes were prepared by a modified SCR performed at a low salt concentration (L-SCR) and directly introduced into commercial electrocompetent E. coli cells. Since successful electroporation relies on the genetic stability of a chromosome in cells, genetically stable 1 Mb chromosomes were developed according to a portable chromosome format (PCF). Using physically and genetically stabilized chromosomes, the democratization of genome synthetic biology will be greatly accelerated.

Tissue-Based Proteomic Profiling in Patients with Hyperplasia and Endometrial Cancer.

Uterine cancers are among the most prevalent gynecological malignancies, and endometrial cancer (EC) is the most common in this group. This study used tissue-based proteomic profiling analysis in patients with endometrial cancer and hyperplasia, and control patients. Conventional 2D gel electrophoresis, followed by a mass spectrometry approach with bioinformatics, including a network pathway analysis pipeline, was used to identify differentially expressed proteins and associated metabolic pathways between the study groups. Thirty-six patients (twelve with endometrial cancer, twelve with hyperplasia, and twelve controls) were enrolled in this study. The mean age of the participants was 46–75 years. Eighty-seven proteins were significantly differentially expressed between the study groups, of which fifty-three were significantly differentially regulated (twenty-eight upregulated and twenty-five downregulated) in the tissue samples of EC patients compared to the control (Ctrl). Furthermore, 26 proteins were significantly dysregulated (8 upregulated and 18 downregulated) in tissue samples of hyperplasia (HY) patients compared to Ctrl. Thirty-two proteins (nineteen upregulated and thirteen downregulated) including desmin, peptidyl prolyl cis-trans isomerase A, and zinc finger protein 844 were downregulated in the EC group compared to the HY group. Additionally, fructose bisphosphate aldolase A, alpha enolase, and keratin type 1 cytoskeletal 10 were upregulated in the EC group compared to those in the HY group. The proteins identified in this study were known to regulate cellular processes (36%), followed by biological regulation (16%). Ingenuity pathway analysis found that proteins that are differentially expressed between EC and HY are linked to AKT, ACTA2, and other signaling pathways. The panels of protein markers identified in this study could be used as potential biomarkers for distinguishing between EC and HY and early diagnosis and progression of EC from hyperplasia and normal patients.

Hapten-labeled fusion-polymerase chain reaction of multiple marker genes for the application of immunochromatographic test

A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive inspection systems. Among these NATs, fusion-PCR (also called as overlap-extension-PCR) has been focused on this study and adopted to generate the fused amplicon composed of plural marker gene fragments for detection. Generally, conventional agarose gel electrophoresis followed by gel staining is employed to check the PCR results. However, these are time-consuming processes that use specific equipment. To overcome these disadvantages, the immunochromatographic test (ICT) for the detection of PCR amplicons with hapten-labels that were generated by PCR using hapten-labeled primers was also adopted in this study. Based on these concepts, we constructed the systems of hapten-labeled fusion-PCR (HL-FuPCR) followed by ICT (HL-FuPCR-ICT) for the two and three marker genes derived from pathogenic microbe. As a result, we successfully developed a two marker genes system for the pathogenic influenza A virus and a three marker genes system for the penicillin-resistant Streptococcus pneumoniae. These detection systems of HL-FuPCR-ICT are characterized by simple handling and rapid detection within few minutes, and also showed the results as clear lines. Thus, the HL-FuPCR-ICT system introduced in this study has potential for use as a user-friendly inspection tool with the advantages especially in the detection of specific strains or groups expressing the characteristic phenotype(s) such as antibiotic resistance and/or high pathogenicity even in the same species.

Genetic diversity of Listeria monocytogenes strains contaminating food and food producing environment as single based sample in Italy (retrospective study)

Human listeriosis outbreaks are often associated with food products, which could be contaminated, at the same time, also by different clones of Listeria monocytogenes. This emphasize the need to type more than one L.monocytogenes isolate found in a single food or environmental sample.The purpose of the present study was to evaluate the presence of different L.monocytogenes strains in food and food production environment in order to understand if there is need to type more isolates from the same sample in case of presence of L.monocytogenes. Between 2011 and 2015, at the Italian National Reference Laboratory for L.monocytogenes, for each positive sample, from two to twenty-three isolates of L.monocytogenes were collected. All the isolates were characterized by conventional serotyping and pulsed field gel electrophoresis (PFGE). Moreover, isolates from the same sample, having indistinguishable PFGE profile, were subjected to whole genome sequencing in order to perform core genome Multi Locus Sequence Typing (cgMLST).Within each sample, more than one serotype and one pulsotype were found in 11.9% and 27.5%, respectively. For indistinguishable PFGE patterns the cgMLST analysis showed 96.2% of concordance demonstrating the added value of new sequencing technologies.This study has demonstrated the need to select and type more than one L.monocytogenes colony in one food or food environmental sample to detect the diversity of L.monocytogenes strains and facilitate downstream investigations and effective source attribution in foodborne outbreak inquiry.

Immunoglobulin D Multiple Myeloma with a “Hidden” Lambda Light Chain

Abstract Introduction/Objective In rare cases, the conventional immunofixation gel electrophoresis technique fails to detect the light chain of an M-protein. We report a case of immunoglobulin (Ig) D multiple myeloma with a hidden lambda (λ) light chain. Methods/Case Report Capillary electrophoresis (CE) (Sebia CAPILLARYS 2) was used to detect and quantify M- proteins in serum specimens. Immunosubtraction (IS) on the CAPILLARYS 2 systems was used to identify the classes of M-proteins. Conventional gel immunofixation electrophoresis (IFE) was performed, using monospecific antisera for IgD, IgE, kappa (κ) or λ in the Sebia HYDRASYS system, and IgG, IgA, IgM, κ or λ in the Helena SPIFE3000 system. Beta-mercaptoethanol (BME) with Fluidil were used as reduction agents. Results (if a Case Study enter NA) Results of serum CE showed two abnormal peaks in beta 2 and gamma regions, suspected to be positive for M-proteins. IS results showed subtraction for λ light chain only in both peaks, suggesting two monoclonal λ light chains. In contrary, no monoclonal λ light chain was detected in gamma region by IFE (Sebia). Epitope masking in the folded monoclonal protein was suspected to cause the “hidden λ light chain” and was further investigated by two laboratory approaches. IFE performed on the Helena SPIFE3000 system found two λ bands in beta 2 and gamma regions, which was consistent with the results from IS. The treatment of BME with Fluidil helped unmasking the hidden epitope and revealed the λ band in gamma region on IFE (Sebia). Conclusion The medical laboratories should be aware of the described scenario. The failure to detect light chains of certain intact M-proteins is most likely due to the structurally inaccessibility of light chains. It is recommended that treatment with reduction agents or use of an alternative methodology or IS might be helpful for investigating suspected heavy chain only cases, due to the limitation of conventional methodology.

A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases

Clustered regularly interspaced short palindromic repeats (CRISPR)−CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. Here, we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small- to large-scale genome editing in plants. This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs (gRNAs) (FLASH), and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time. The identities of gRNAs in Agrobacterium mixtures and transgenic plants can therefore be read out by detecting the FLASH tags, a process that requires only conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting all 1,072 members of the receptor-like kinase (RLK) family in rice. One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs, and 74.3% (710/955) of the target genes had three or more independent T0 lines. Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly (92.1%) associated with frameshift mutations in the target genes. In addition, the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes. Furthermore, we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes. This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics.