Positive Plasmid Research Articles

Complete sequences of two novel blaNDM-1-harbouring plasmids from two Acinetobacter towneri isolates in China associated with the acquisition of Tn125

Two novel New Delhi metallo-β-lactamase-1 (NDM-1)-positive plasmids containing a complete composite transposon, Tn125, from two respective Acinetobacter towneri isolates were characterized. Plasmid pNDM-GJ01 (30,293 bp) isolated from A. towneri G165 did not show homology to any known plasmid structure, except for the transposon Tn125 containing blaNDM-1. A novel repB gene and two XRE-type transcriptional regulators were found in pNDM-GJ01. Plasmid pNDM-GJ02 (62,011 bp) isolated from A. towneri G295 showed the highest homology to pBJAB0715 (41% coverage, 99% nucleotide identity). In addition to the blaNDM-1-harbouring transposon Tn125, pNDM-GJ02 also had an IS26-composite transposon, which contains ISCR1 and two class 1 integrons carrying different cassette arrays. Both clinical isolates were highly resistant to β-lactams and susceptible to tigecycline and colistin. Ten other resistance genes were detected in G295, and one other resistance gene was detected in G165. No transconjugant was obtained from any of the donors by broth and filter mating. The emergence of these two novel plasmids carrying NDM-1 in Acinetobacter spp., pNDM-GJ01 and pNDM-GJ02, suggests Tn125 mobile integration.

Establishment and application of the molecular-beacon-based asymmetric recombinase amplification for detecting Staphylococcus aureus

Objective To establish a homothermal and fast detecting method on pathogenic bacteria by combining recombinase-aid amplification (RAA) with molecular beacon. Methods The establishment of the methodology. Staphylococcus aureus specific primers were designed from the relative region of the staphylococcal protein A (SPA). Asymmetry amplification was optimized by adjusting the primer concentration ratios. The results of amplification and hybridization were visualized and analyzed by agarose gel electrophoresis and fluorescence detection. The sensitivity was identified by detecting dilute positive plasmids. And the specificity was determined using RAA method by detecting 72 pathogenic bacteria, including Staphylococcus aureus and other Staphylococcus spp. from the Department of Clinical Laboratory of Daping Hospital in December 2016. Besides, the Kappa analysis and the clinical diagnosis efficiency were investigated by analyzing 39 extra strains in the laboratory in December 2016. Results When the concentration ratio of restrictive and non-restrictive primer was 1∶20, the yield efficiency of single-stranded DNA (ssDNA) reached the peak. And as for the hybridization efficiency, the asymmetry amplification was higher than symmetry amplification. Twenty copies/μl was proposed as the limits of detection by testing dilute plasmids. And the RAA hybridization method could distinguish Staphylococcus aureus with other Staphylococcus spp.Comparing with traditional detection methods with a Kappa index of 0.860, this method shows a good consistency. By analyzing the 111 bacteria, the sensitivity of the method is 92.5% (37/40), the specificity is 97.2% (69/71), the positive predictive value is 94.9% (37/39), the negative predictive value is 95.8% (69/72), the positive likelihood radio is 33.04, the negative likelihood radio is 0.077, the Youden index is 0.897 and the Kappa index is 0.902. Conclusion Through the combination of asymmetry recombinase-aid amplification optimization and molecular beacon probe, a new method of detecting bacteria DNA with RAA hybridization technique is established, providing the foundation for its clinical application. (Chin J Lab Med, 2017, 40: 309-313) Key words: Staphylococcus aureus; Nucleic acid amplification techniques; Molecular probes

Development of pBT63, a positive control plasmid for qualitative detection of genetically modified rice

Development of pBT63, a positive control plasmid for qualitative detection of genetically modified rice

Prokaryotic Expression of α-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia.

To study prokaryotic expression and subcellular localization of α-13 giardin in Giardia lamblia trophozoites, α-13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into E. coli BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of α-13 giardin was determined by fluorescence immunoassay. The results showed that the length of α-13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti-α13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant α-13 giardin. Immunofluorescence assays revealed that α-13 giardin was localized in the cytoplasm of G. lamblia trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on α-13 giardin of G. lamblia.

Development and Application of a Quantitative Real‐time Polymerase Chain Reaction Assay for the Detection of Aeromonas salmonicida

AbstractA rapid, economical, specific, and sensitive quantitative real‐time polymerase chain reaction (qPCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Aeromonas salmonicida from farmed Atlantic salmon, Salmo salar, with the symptoms of furunculosis. The set of primers designed from the virulence array protein (vapA) gene was specific to A. salmonicida. Compared with the conventional PCR, qPCR had a lower detection limit of 5.6 copies of the positive plasmids. The standard curve, which showed the relationship between the copies of A. salmonicida and its quantification cycle (Cq) value, could be described as follows: log (copies of A. salmonicida) = −0.3213 Cq + 10.721. The quantitative detection of copies of A. salmonicida in different tissues of the moribund Atlantic salmon showed that A. salmonicida could be detected in all tissues; the spleen contained the largest number of A. salmonicida and then the kidney. These results suggest that the qPCR assay reported here is a specific, sensitive, and quantitative method for detecting A. salmonicida. It can be used for the routine tests of A. salmonicida in local aquaculture enterprise and for the research of infection routes of A. salmonicida to Atlantic salmon.

Development and Optimization of a Reverse Transcription Hemi-Nested PCR Primer for the Detection of Potato Mop-Top Virus at Quarantine Inspection Sites in Korea.

Potato mop-top virus (PMTV) is classified as a plant quarantine virus in Korea. It is tested at import inspection sites. In this study, two sets of reverse transcription-hemi-nested polymerase chain reaction primers were developed to diagnose PMTV. In addition, modified- positive control plasmid was developed for the identification of false positive results in plant quarantine. The assay is expected to be useful for improving the detection and diagnosis of PMTV.

Immunolocalization of α18- and α12-giardin in Giardia lamblia trophozoites.

To study subcellular localization of α18- and α12-giardin in Giardia lamblia trophozoites, the α18- and α12-giardin genes were amplified from G. lamblia assemblage A, respectively. The PCR products were cloned into the prokaryotic expression vector pET-28a(+), and the positive recombinant plasmids were transformed into E. coli Rosetta (DE3) strain for the expression, and expressed α18- and α12-giardin fusion protein were purified by Ni-Agarose resin, respectively. Mice were immunized with purified fusion proteins for preparation of polyclonal antibody, and then the subcellular localization of α18- and α12-giardin was determined by fluorescence immunoassay. Results showed that the concentrations of purified α18- and α12-giardin fusion proteins were 1.20 and 0.86mg/ml, respectively. The titers of anti-α18- and anti-α12-giardin polyclonal antibody were both as high as 1:25600 dilutions. Immunofluorescent analysis showed that α18- and α12-giardin proteins were mainly localized at four pairs of flagella and the cytoplasm of G. lamblia trophozoites, suggesting that α18- and α12-giardin are the flagella and cytoplasm-associated proteins, respectively. The above information would lay the foundation for research about the crystal structure and biological function of α18- and α12-giardin.

Abstract 1376: Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency (<0.1%) was also detected in the same reaction. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for exon19 deletion, and 0.9959 for T790M, respectively. CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1376.

Molecular structure and alternative splicing analysis of heat shock factors of Schistosoma japonicum

To clone and identify the heat shock factors (HSFs) of Schistosoma japonicum and analyze its molecular structure and alternative splicing pattern. The New Zealand rabbits were infected with the cercariae of Schistosoma japonicum and were killed and dissected 42 days post-infection, and the adult worms of S. japonicum and the livers of the rabbits were harvested. Then, the total RNA was extracted by using Trizol reagent. The Sj-hsf open reading frame (ORF) and the alternative splicing fragments were amplified by RT-PCR from the female, male and egg samples, then cloned and verified by enzyme digestion and sequencing. DNAMAN 8.0, InterPro, Mega 6 combined with the Internet databases were utilized to clarify the gene structure, functional domains, alternative splicing pattern, and the homology and phylogenetic tree of HSFs. Sj-hsf ORF and the alternative splicing fragments were amplified from the female, male and egg samples of S. japonicum by RT-PCR. After cloning, the positive recombinant plasmids pBSjHSFf-F, pBSjHSFf-M, pBSjHSFf-E containing Sj-hsf ORF, pBSjHSFs-F, pBSjHSFs-M, pBSjHSFs-E with Sj-hsf alternative splicing fragments were identified by enzyme digestion and sequencing. Three alternative splicing Sj-hsf isoforms were observed through sequence analysis: Sj-hsf-isoform1 (2 050 bp), Sj-hsf -isoform2 (2 086 bp) and Sj - hsf -isoform3 (2 111 bp); the GenBank accession numbers were KU954546, KX119143 and KX119144, respectively. All the three isoforms located in the same Contig SJC_S000780 of S. japonicum genome and all expressed at female, male and egg stages, but Sj-hsf-isoform1 with a high-level expression. Sj-HSF-isoform1 (671 aa) and Sj-HSF-isoform2 (683 aa) had DBD (DNA binding domain), HR-A/B and HR-C domains, while Sj-HSF-isoform3 (282 aa) stopped in advance without HR-C domain. Phylogenetic tree analysis of HSFs illustrated that Sj - HSFs belonged to HSF1 family, with a close phylogenetic relationship to Sm-HSFs. There are three alternative splicing isoforms of Sj-HSF existing in the female, male and egg stages of S. japonicum, but Sj-HSF-isoform1 expresses in a high-level. This study lays the foundation for further study on molecular mechanisms of Sj-HSFs in regulating the heat shock response system.

Transforming growth factor beta 1 induced endothelin-1 release is peroxisome proliferator-activated receptor gamma dependent in A549 cells.

BackgroundEndothelin-1 (ET-1) is involved in pulmonary vascular remodeling. The aim of this study was to investigate the biochemical interactions between PPAR-γ, TGF-β1 and ET-1 in vitro.MethodsA549 cells were pre-treated with S2505 (10 μM), S2871 (10 μM) with/without SB203580 (10 μM) for 60 min following 2 h treatment with 10 ng/mL TGF-β1. A549 cells were also transfected with positive or negative PPAR-γ plasmids for comparison. RT-PCR, ELISA, western blotting and confocal laser scanning microscopy (CLSM) were used to measure the relevant expression of mRNA, protein, mediators of pathways and nuclear factor translocation.ResultsSB203580 inhibited TGF-β1 induced ET-1 expression in A549 cells. S2871 decreased PPAR-γ mRNA and increase TGF-β1-induced ET-1 expression. S2871 increased phosphorylation of p38 MAPK and Smad2. Cells transfected with PPAR-γ negative plasmid increased TGF-β1 induced ET-1 expression, and increased the expression of phospho-p38 MAPK and phospho-Smad2. S2505 increased PPAR-γ mRNA expression, suppressed the increased TGF-β1-induced expression of ET-1. S2505 inhibited TGF-β1 induced phosphorylation of p38 MAPK and Smad2, also the nuclear translocation of Smad2. Cells transfected with PPAR-γ positive plasmid reduced TGF-β1-induced ET-1 expression, and inhibited the expression of phospho-p38 MAPK and phospho-Smad2.ConclusionsTGF-β1 induced release of endothelin-1 is PPAR-γ dependent in cultured A549 cells.

Development and validation of a new real-time assay for the quantification of Verticillium dahliae in the soil: a comparison with conventional soil plating

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope = −3.405, r2 = 0.976, E = 96.64 %) and the positive recombinant plasmid (y = −3.36, r2 = 0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 102 copies/μL (CV% ≈ 1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g−1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r2 = 0.944) and with the soil plating method (r2 = 0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.

Detection of Echinococcus granulosus and Echinococcus multilocularis in cyst samples using a novel single tube multiplex real-time polymerase chain reaction

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical findings, are being used. The definitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RT-PCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') and Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primers and three different probes; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC_000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confirmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M-RT-PCR. E.granulosus and E.multilocularis control plasmids were constructed to detect analytic sensitivity of the test using TOPO cloning. Positive control plasmids were diluted to determine analytical sensitivity and specificity by distilled water at 10(6)-10(5)-10(4)-10(3)-10(2)-10(1)-1 plasmid copy of dilution in each reaction. According to the results, analytical sensitivity of the assay for E.granulosus and E.multilocularis was 1 copy plasmid/µl reaction. The non-existence of cross reactivity with 12 different parasites' DNA samples showed the analytical specificity of the assay. Displaying Echinococcus DNA in cyst samples among 25 patients and species discrimination as well as non-existence of cross reactivity with human DNA samples showed that the clinical sensitivity and specificity of the assay were 100%. As a result, the M-RT-PCR developed in the present study provided a sensitive, specific, rapid, and reliable method in the diagnosis of echinococcosis and the discrimination of E.granulosus and E.multilocularis from cyst samples.

Simultaneous detection of Waddlia chondrophila and Listeria monocytogenes in aborted ruminant samples by real-time quantitative PCR

Waddlia chondrophila and Listeria monocytogenes are well known emerging pathogens that cause ruminants' abortion around the world. Zoonotic infections caused by these bacteria are mostly underestimated due to difficulties of diagnosis resulting from their intracellular growth. The purpose of this study was to develop and validate a dual real-time quantitative PCR (qPCR) assay for the simultaneous quantification of W. chondrophila and L. monocytogenes in biological samples from aborted ruminants. Technical performance was examined using linear control plasmids. A total of 211 veterinary samples (15 placental tissues, 50 blood, 53 milk and 93 vaginal swab samples) were used to compare the qPCR with standard culture methods. The limit of detection was 2 plasmid template copies per reaction with approximately 5000-fold differences in concentrations of the two competing templates. Coefficients of variation for positive control plasmids were less than 1.8%. Both intra- (1.01–2.13% and 1.13–1.71%) and inter- (1.02–2.24% and 1.2–1.66%) assay variations of qPCR for W. chondrophila and L. monocytogenes plasmids were within the acceptable limits, implying high reproducibility and repeatability of the assay. The use of the qPCR assay resulted in 53 positive identifications among 211 veterinary samples against only 19 when standard cultural methods were used. On the basis of these results, we determined 100% sensitivity and 100% specificity for our new qPCR. Dual abortigenic agents were identified in 8.6% and 26.6% of vaginal swab samples and placental tissues, respectively. To conclude, this new qPCR will be of great value in the simultaneous and rapid diagnosis of W. chondrophila and L. monocytogenes in large-scale screening programs and also during outbreaks.

Rapid Screening of Recombinant Plasmids by Direct Colony Quantitative Real-Time PCR

Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.

Sequence analysis and prokaryotic expression of Giardia lamblia α-18 giardin gene

To study the genetic variation and prokaryotic expression of α18 giardin gene of Giardia lamblia zoonotic assemblage A and host-specific assemblage F, the α18 genes were amplified from G. lamblia assemblages A and F by PCR and sequenced. The PCR product was cloned into the prokaryotic expression vector pET-28a(+) and the positive recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) strain for the expression. The expressed α18 giardin fusion protein was validated by SDS-PAGE and Western blot analysis, and purified by Ni-Agarose resin. The putative sequence of α18 giardin amino acid was analyzed by bioinformatics software. Results showed that the α18 giardin gene was 861bp in length, encoding 286 amino acids; it was 100% homologous between human-derived and dog-derived G. lamblia assemblage A, but it was 86.8% homologous with G. lamblia assemblage F (cat-derived). Giardin α18 was about 36kDa in molecular weight, with good reactivity. Prediction based on in silico analyses: it had hydrophobicity, without signal peptide and transmembrane domain, and contained 11 alpha regions, 13 beta sheets, 1 beta turn and 7 random coils in secondary structure. The above information would lay the foundation for research about the subcellular localization and biological function of α18 giardin in G. lamblia.

Abstract 4908: Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of EGFR tyrosine kinase inhibitor (TKI) treatment. Three most common EGFR mutations (L858R, exon19 deletion, and T790M) are known to be major selection marker for EGFR-TKI therapy. Noninvasive techniques for tumor genotyping may be needed to fully realize the potential of genotype-directed cancer care as a “Liquid biopsy”. The low abundance of mutants and limited amount and quality of available cell-free DNA (cfDNA) in plasma samples render it difficult to reliably detect multiple mutations with current platforms and methods. Here, we develop a multiplex dPCR assay to detect three common EGFR mutations in one reaction using picodroplet digital PCR (ddPCR) technology, which enable us to provide 3-fold throughput and save the cost as well as DNA samples. METHODS: Positive and negative control plasmids for the EGFR assay were prepared by cloning DNA fragments containing wild-type or the EGFR mutations. The appropriate concentration of plasmid DNA was determined empirically to yield a mixture in which the number of copies of mutant DNA was ca. 0.01-1.00% of the number of wild-type EGFR fragments. Genomic DNA from the lung tumor cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. RESULTS: Serial dilutions experiments using plasmid as well as genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9983for L858R, 0.9911 for exon19 deletion, and 0.9781 for T790M. The limit of blank (LOB) was defined by the frequency of positive droplets measured in 400 ng of wild-type DNA control samples. The number of false events of mutant droplets detected per analysis is: 8 for L858R, 6 for Exon 19 deletion, 6 for T790M from human normal gDNA, and 9 for L858R, 4 for Exon 19 deletion, 2 for T790M from A549 gDNA. Thus, the LOB was determined as a 10 events/assay. CONCLUSIONS: Using ddPCR, we established multiplex and ultra-sensitive genotyping platform for three common EGFR mutations. Results of a proof-of-principle study using preclinical model indicated that the clinical utility of multiplex ddPCR to detect for multiple EGFR mutations. Further evaluation using clinical samples is warranted. Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Masaru Watanabe, Takashi Kikuchi, Masanori Nakanishi, Kazuto Matsunaga, Nobuyuki Yamamoto. Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4908. doi:10.1158/1538-7445.AM2015-4908

Comparing the multiplex RT-PCR method and liquichip technology in the detection of diarrhea-related virus

Objective To compare the detection efficiency between multiplex RT-PCR method and liquichip technology for screening the viral etiological agents of diarrhea. Methods The development of the multiplex RT-PCR method. A total of 107 feces samples from patients who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from September 2013 to February 2014 were collected and tested in parallel by both multiplex RT-PCR and xTAG Gastrointestinal Pathogen Panel (xTAG GPP) for Adenovirus, Norovirus genogroupⅠandⅡ, as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus. To evaluate the sensitivity and specificity of multiplex RT-PCR, xTAG GPP and monoplex RT-PCR were used as reference.Kappa coefficient test was used to evaluate the consistency among the methods. The detection limit and accuracy of multiplex RT-PCR were evaluated by detection of serial dilution of positive plasmids and products sequencing for the five viral agents. Results The multiplex RT-PCR showed high consistency with xTAG GPP and monoplex RT-PCR, in which Kappa value was 0.885 and 1.000 respectively(P=0.000). Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were at average of 80.8%(21/26) and 100%(295/295)respectively. The detection limit and accuracy of multiplex RT-PCR were 104copies /μl-106copies/μl. Conclusion The high consistency indicated that both the multiplex RT-PCR and xTAG GPP are useful as a special, sensitive, high throughput and rapid diagnostic tools for the detection of the major viral pathogens related to diarrhea in clinical laboratory. (Chin J Lab Med, 2015, 38: 387-391) Key words: Diarrhea; Adenoviridae; Norovirus; Mamastrovirus; Multiplex polymerase chain reaction; Reverse transcriptase polymerase chain reaction

Antibody study in canine distemper virus nucleocapsid protein gene-immunized mice.

The gene for the nucleocapsid (N) protein of canine distemper virus was cloned into the pMD-18T vector, and positive recombinant plasmids were obtained by enzyme digestion and sequencing. After digestion by both EcoRI and KpnI, the plasmid was directionally cloned into the eukaryotic expression vector pcDNA; the positive clone pcDNA-N was screened by electrophoresis and then transfected into COS-7 cells. Immunofluorescence analysis results showed that the canine distemper virus N protein was expressed in the cytoplasm of transfected COS-7 cells. After emulsification in Freund's adjuvant, the recombinant plasmid pcDNA-N was injected into the abdominal cavity of 8-week-old BABL/c mice, with the pcDNA original vector used as a negative control. Mice were immunized 3 times every 2 weeks. The blood of immunized mice was drawn 2 weeks after completing the immunizations to measure titer levels. The antibody titer in the pcDNA-N test was 10(1.62 ± 0.164), while in the control group this value was 10(0.52 ± 0.56), indicating that specific humoral immunity was induced in canine distemper virus nucleocapsid protein-immunized mice.

Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry

The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

Development of a PCR Diagnostic System for Iris yellow spot tospovirus in Quarantine.

Iris yellow spot virus (IYSV) is a plant pathogenic virus which has been reported to continuously occur in onion bulbs, allium field crops, seed crops, lisianthus, and irises. In South Korea, IYSV is a “controlled” virus that has not been reported, and inspection is performed when crops of the genus Iris are imported into South Korea. In this study, reverse-transcription polymerase chain reaction (RT-PCR) and nested PCR inspection methods, which can detect IYSV, from imported crops of the genus Iris at quarantine sites, were developed. In addition, a modified positive plasmid, which can be used as a positive control during inspection, was developed. This modified plasmid can facilitate a more accurate inspection by enabling the examination of a laboratory contamination in an inspection system. The inspection methods that were developed in this study are expected to contribute, through the prompt and accurate inspection of IYSV at quarantine sites to the plant quarantine in South Korea.