Determination of honey adulterated with corn syrup by quantitative amplification of maize residual DNA using ultra-rapid real-time PCR.

BACKGROUND: Honey is a naturally sweet syrup made by honeybees from floral nectar. However, high-fructose corn syrup has been prevalently used for the adulteration of honey. A novel molecular method was developed for the characterization of corn syrup-adulterated honey by specific amplification and quantification of maize residual DNA in honey. An ultra-rapid real-time polymerase chain reaction (UR-qPCR) system for rapid amplification and protocol for direct purification of residual DNA from honey were described. RESULTS: Rapidity of maize DNA amplification was acquired within 20 min for a limit of detection of around three copies of targeted DNAs. The amplification of maize residual DNA in honeys adulterated with corn syrup from 5% to 80% (v/v) showed that a minimum rate of 10% adulteration can be identified, and Maize genomic DNA in 5 mL of adulterated honeys was from 13 ± 9 copies to 2478 ± 827 copies, respectively. However, the residual DNA of maize was also detected in natural honey produced in the region where pollen and nectar of maize were collected, and the quantity of maize genomic DNA in these natural honeys was in the range of 10% adulteration with corn syrup. Therefore, detection of both pollen and residual DNA of maize in honey is important in identifying the source of maize residual DNA present in honey. CONCLUSION: A rapid PCR assay was first developed for the accurate detection and quantification of maize residual DNA in honey. It is a useful tool for specific identification of the corn syrup used for honey adulteration. Further studies on residual DNA in various types of corn syrup and specificity of primer are recommended. © 2021 Society of Chemical Industry.

Interactions between lysophosphatidylinositol receptor GPR55 and sphingosine-1-phosphate receptor S1P5 in live cells.

Lysophosphatidylinositol (LPI) and sphingosine-1-phosphate (S1P) are bioactive lipids implicated in various cellular events including proliferation, migration, and cancer progression. LPI and S1P act as ligands for G-protein coupled GPR55 and S1P receptors, respectively, and activate specific signaling pathways. Both receptors are highly expressed in various cancer tissues and associated with tumor progression. However, physical and functional crosstalk between the two receptors has not been elucidated to date. Bioluminescence resonance energy transfer (BRET) experiments in the current study showed that S1P5 strongly and specifically interacts with GPR55. We observed co-internalization of both receptors upon agonist stimulation. Notably, activation of one receptor induced co-internalization of the partner receptor. Next, we examined functional crosstalk of the two receptors. Interestingly, while activation of the individual receptors augmented cell proliferation, ERK phosphorylation and cancer-associated gene expression in HCT116 cells, co-activation of both receptors inhibited these stimulatory effects. Our collective findings indicate that GPR55 and S1P5 form a heterodimer and their co-activation attenuates the stimulatory activity of each receptor on colon cancer progression.

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR.

BACKGROUND: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. OBJECTIVES: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. METHODS: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. RESULTS: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 104 spores/bee, and the stable detection level was ≥ 2.40 × 105 spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 104 copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. CONCLUSIONS: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip.

Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site.

Application of ultra-rapid qPCR and DNA chips for viral RNA detection and confirmation.

The rapid and accurate detection of the presence of microorganisms, such as viruses, has been an important issue in the fields of public health, as well as agriculture. A PCR-based detection method has been developed and applied in these fields to determine the presence of specific pathogens. Although the major advantage of real-time PCR is the monitoring of amplification and ability to quantify the template genes, the method described here should solve the problem of nonspecific product synthesis. We obtained viral RNA from infected samples by freezing and thawing; we rapidly synthesized cDNA from RNA, and then amplified the cDNA by rapid PCR in 10 Min. Finally, the PCR products were hybridized and quickly confirmed to be the target analyte on a DNA chip. Our newly proposed methods overcome the drawbacks of PCR-based detection and provide three additional advantages, namely, rapidly obtaining large amounts of RNA from samples, quickly detecting infective or pathogenic genes, and speedily confirming the detected exogenous genes. This application might be useful for detecting viral RNA and for the diagnosis of RNA virus-mediated diseases.

Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis.

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×104 CFU/mL and 2.0×106 CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Analysis of the complete genome sequence and capsid region of black queen cell viruses from infected honeybees (Apis mellifera) in Korea.

Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97-100% identity. They also showed 92-99% similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4-6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92%) with Hungary10, but low similarity (86%) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5'-UTR, the intergenic region, and the capsid regions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.

Phylogenetic analysis of black queen cell virus genotypes in South Korea.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Reverse transcription loop-mediated isothermal amplification for sensitive and rapid detection of Korean sacbrood virus.

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 10(3) copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees.

Rapid detection of sacbrood virus in honeybee using ultra-rapid real-time polymerase chain reaction.

A real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for the fast and highly sensitive detection of the sacbrood virus (SBV) genome and applied to honeybee samples. Using plasmid DNA containing a partial SBV genome and diluted serially, as few as 1×10(2)copies/µl (correlation co-efficiency >0.99) were detected by the qRT-PCR assay, whereas 1×10(3)copies/µl were detected by the conventional RT-PCR assay. As a rapid detection method, ultra-rapid real-time PCR (URRT-PCR) was carried out with a GenSpector TMC-1000 silicon-glass chip-based thermal cycler, which has a 6µl micro-chamber volume and a fast outstandingly heating/cooling rate. Using this method, 10(3)copies of pBX-SBV3.8 clone were detected within 17 min after 40 PCR cycles, including melting point analysis. To reduce the detection time for SBV, synthesis of the cDNA of the SBV genome from a honeybee sample was attempted for different reaction times and the cDNA was used as the template for URRT-PCR assays. The results indicated that a 5 min reaction time was sufficient to synthesize cDNA as the template for the SBV URRT-PCR assay. This study described a novel PCR-based method that is able to detect an RNA virus in environmental samples within 22 min, including reverse transcription, PCR detection and melting point analysis in real-time.

Development of a rapid detection method to detect tdh gene in Vibrio parahaemolyticus using 2-step ultrarapid real-time polymerase chain reaction.

Thermostable direct hemolysin encoded by tdh gene has been considered an important virulence factor in pathogenic Vibrio parahaemolyticus. Two-step ultrarapid real-time polymerase chain reaction (URRT PCR) with a microchip was devised to detect V. parahaemolyticus carrying tdh gene. This novel method has a 6-µL reaction volume and extremely reduces running time since one cycle can be completed in 10 s or less. Consequently, 35 cycles of URRT PCR was successfully able to detect up to 100 fg (18 copies) of genomic DNA from pathogenic V. parahaemolyticus carrying tdh gene in 6 min. These results indicate that this method is at present the most rapid detection method for tdh gene and pathogenic V. parahaemolyticus.

Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli using two-step ultra-rapid real-time PCR.

A rapid detection method for Enterohemorrhagic Escherichia coli (EHEC), which has the virulent stx2 gene, was developed using a two-step, ultra-rapid real-time (URRT) PCR. URRT PCR was designed to detect the stx2 gene using a microchip-based, real-time PCR system, GenSpector TMC-1000, which only has a 6 microl total reaction volume with an extremely short denaturation step and combined annealing/extension step (1 and 3 s, respectively) for each cycle. Specific primers for the stx2 gene were designed to amplify a 100 bp region known for genetic stability among the various EHEC strains. Using the URRT PCR method, stx2 gene could be detected in 7 min 8 s including melting point (Tm) analysis. The detection limit for the stx2 gene for URRT-PCR was estimated to be 3 c.f.u./PCR with the amplification product having a consistent Tm of 85.2 +/- 0.4 degrees C. This method was tested for the various applications relevant to the different EHEC strains and was useful for the rapid detection of stx2-carrying EHEC strains.

Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB).

A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene of P. larvae with a micro-scale chip-based real-time PCR system, GenSpector TMC-1000, which has uncommonly fast heating and cooling rates (10 degrees C per second) and small reaction volume (6microl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools.

Chinese cabbage extracts and sulforaphane can protect H2O2-induced inhibition of gap junctional intercellular communication through the inactivation of ERK1/2 and p38 MAP kinases.

The cruciferous vegetables such as Chinese cabbages and broccoli are known to have anticancer phytochemicals, and the consumption of cruciferous vegetables has been proposed to protect against various cancers. The anticarcinogenic properties of some Chinese cabbage extracts and sulforaphane glucosinolate (SFN) were assessed by examining their ability to prevent the inhibition of gap junctional intercellular communication (GJIC) induced by hydrogen peroxide (H2O2) in WB-F344 normal rat liver epithelial cells. The cells were preincubated with Chinese cabbage extracts and SFN for 24 h followed by cotreatment with cells and H2O2 (750 microM) for 1 h. Chinese cabbage extracts and SFN prevented the inhibition of GJIC and phosphorylation of gap junction protein connexin43 (Cx43) by H2O2 treatment. Chinese cabbage extracts and SFN were able to prevent the inhibition of GJIC through the blocking of Cx43 phosphorylaton and inactivation of ERK 1/2 and p38 MAP kinase. The results suggest that cruciferous vegetables and their components, SFN, may exert the anticancer effect by targeting the GJIC as a functional dietary chemopreventive agent.

Role of gap junctional intercellular communication (GJIC) through p38 and ERK1/2 pathway in the differentiation of rat neuronal stem cells.

Gap junctional intercellular communications (GJIC) contributes to neural function in development and differentiation of CNS. In this study, we have investigated the expression of GJIC during the differentiation of neuronal stem cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neuronal stem cell-derived cells from rat brain. During neuronal stem cell differentiation, expressions of Cx43 and 32 were increased for the duration of 72 hr, however the effect were decreased on the 7d. In the neuronal stem cell-derived cells, pretreatments with p38 MAP kinase inhibitor, SB203580, and MEK inhibitor, PD98059, could protect GJIC against TPA-induced inhibition of GJIC. Our data suggest that GJIC plays an important role during neuronal stem cell differentiation, and ERK1/2 and p38 MAP kinase signaling pathway may be closely related functionally to regulate gap junction in rat neuronal stem cell-derived cells.

Development of a sandwich ELISA for the detection of Listeria spp. using specific flagella antibodies.

Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x 10(5) cells/0.1 ml at 22 degrees C. and 1 x 10(6) cells/0.1 ml at 30 degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 10(4) cells/0.1 ml at 22 degrees C and 30 degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.

Polymerase chain reaction analysis of eae gene subtypes present in attaching and effacing Escherichia coli isolated from pigs with diarrhea.

In this study the subtype of eae gene was determined by polymerase chain reaction for a total of 59 attaching and effacing Escherichia coli isolated from preweaned (38 isolates) and postweaned (21 isolates) pigs. The eae(beta) gene detected in 19 E. coli from preweaned pigs and 10 E. coli from postweaned pigs was found to be the most common subtype, followed by eae(gamma), eae(epsilon), and eae(zeta) genes. Subtypes were not determined for 7 E. coli isolates. No other subtype of the eae gene was detected in eae+ E. coli evaluated in this study.

Mutations induced by 1,3-butadiene metabolites, butadiene diolepoxide, and 1,2,3,4-diepoxybutane at the Hprt locus in CHO-K1 cells.

Butadiene (BD) is an important industrial chemical that is classified as a probable human carcinogen. Butadiene diolepoxide (BDE) and 1,2,3,4-diepoxybutane (DEB) are metabolites of carcinogenic BD and contain the DNA-reactive one and two epoxides, respectively. In this study, the mutation frequencies and mutation spectra that are induced by BDE and DEB have been investigated at the hprt locus in CHO-K1 cells. The BDE- and DEB-treated CHO-K1 cells were allowed to grow for several days, then seeded in a medium that contained 6-thioguanine in order to select the hprt mutants. BDE exhibited the mutagenic activity at concentrations that were approximately 100-times higher than DEB. The mutation spectra for BDE and DEB were determined by a reverse transcription-polymerase chain reaction of hprt mRNA, which was followed by automatic DNA sequencing of the PCR products. The mutational spectrum for BDE was exon deletions (16/41), G x C --> A x T transitions (11/41), and A x T --> G x C transitions (5/41). The mutational spectrum for DEB was exon deletions (15/39), G x C --> A x T transitions (11/39), and A x T --> T x A transversions (5/39). The most common base substitution that was induced by both BDE and DEB was G x C --> A x T transitions. The sites of the single base substitutions that were induced by BDE and DEB were guanine and adenine, which was consistent with the DNA adduct profiles. The high frequencies of the exon deletions by each metabolite occurred in the regions of exons 2, 3, or 4. These data indicate that BDE and DEB are mutagenic carcinogens by forming DNA adducts at the site of adenine and guanine, and inducing large exon deletions and single base substitutions.