Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study.

Antibody-conjugated ferromagnetic nanoparticles with lateral flow test strip assay for rapid detection of Campylobacter jejuni in poultry samples.

The aim of this study was to develop a nanoparticle-based cell capture system combined with a lateral flow test strip (LFT) assay for rapid detection of Campylobacter jejuni from poultry samples. The developed assay was bench-marked against the standard modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) method according to ISO16140:2003 procedures. The synthesized ferromagnetic nanoparticles (FMNs) were modified with glutaraldehyde, then functionalized with polyclonal antibodies for specific C. jejuni capture and concentration from poultry samples. After lysing captured cells, DNA from C. jejuni was amplified by PCR using the primers designed to target the hipO gene, and the PCR amplicons were detected with the lateral flow test strip assay. Following the ISO16140:2003 guidelines, the relative detection limit, and the inclusivity and exclusivity tests were determined. The results showed that the limit of detection (LOD) of the assay was 100 or 1 cfu/ml with C. jejuni in pure culture and 101-102 cfu/ml with target cells spiked in poultry sample. In addition, the inclusivity and exclusivity tests were found to be 100%. Using field chicken samples (n = 60), the assay showed relative accuracy, relative specificity, and relative sensitivity of 96.67%, 100% and 93.33%, respectively. The positive predictive values (PPV) and negative predictive values (NPV), and the kappa index of concordance (k) were calculated as 100% and 93.75%, and 0.93, respectively. The developed assay required approximately 3 h to complete and gave results comparable to those analyzed by the standard culture method, which required 5-7 days. The assay is rapid, easy-to-use, and has potential to be directly applied to C. jejuni detection in various categories of poultry samples.

Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification.

The integration of loop-mediated isothermal amplification (LAMP) and DNA-functionalized AuNPs as visual detection probes (LAMP-AuNPs) was developed and applied for the detection of white spot syndrome virus (WSSV) from Penaeid shrimp in this study. The principle of this combination assay relies on the basis of stability characteristics of the DNA-functionalized AuNPs upon hybridization with the complementary target DNA toward salt-induced aggregation. If the detected target DNA is not complementary to the ssDNA probes, the DNA-functionalized AuNPs will be aggregated due to the screening effect of salt, resulting in the change of solution color from red to blue/gray and shift of the surface plasmon peak to longer wavelength. While the DNA-functionalized AuNPs are perfectly matched to the detected target DNA, the color of solution still remains red in color and no surface plasmon spectral shift. This assay provides simply technique, time-saving and its detection results could be achieved qualitatively and quantitatively by visualization using the naked eye due to the colorimetric change and by measurement using the UV-vis spectroscopy due to the surface plasmon spectral shift, respectively. In this study, LAMP-AuNPs assay was successfully developed with the detection of WSSV-LAMP generated product at 0.03 µg/reaction, and showed the sensitivity of 2 × 10(2) copies WSSV plasmid DNA, that is comparable to the most sensitive method reported to date. The LAMP-AuNPs assay described in this study revealed a highly sensitive, rapid and reliable diagnostic protocol for detection of WSSV. This technique has a potential as a routine method for assessing the infectious diseases in Penaeid shrimp not only for WSSV, but also for other shrimp pathogens, and can be useful tool in field conditions for the diagnosis or surveillance programs.

Rapid and sensitive detection of Laem-Singh virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

Laem-Singh virus (LSNV) was discovered recently in Thailand in farmed Giant Tiger shrimp (Penaeus monodon) displaying signs of slow growth syndrome. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here a reverse transcription (RT)-LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect LSNV RNA rapidly and specifically. The reaction was optimized at 65°C for 30 min and amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at LFD test line 5 min after application. Including 10 min for rapid RNA extraction, test results could be generated within 1h and did not require electrophoresis. Compared to an existing RT-PCR method, the RT-LAMP-LFD was also ∼1000-fold more sensitive in detecting LSNV RNA.

Molecular cloning and characterization of two cDNAs encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Hevea brasiliensis.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, EC: 1.1.1.267) is the second enzyme in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, one of the two pathways in plants that can produce isoprenoids. The MEP pathway is the source of isoprene emitted from leaves, but rubber production is believed to result primarily from the mevalonic acid (MVA) pathway. Two cDNAs for DXR designated HbDXR1 and HbDXR2 were isolated from leaves and latex of rubber tree using RT-PCR based methods. Both cDNAs contain an open reading frame (ORF) of 1416bp encoding 471 amino acids with a molecular mass of about 51kDa. The deduced HbDXRs show extensive sequence similarities to that of other plant DXRs (73-87% identity). Molecular modeling revealed that the two HbDXRs contain all typical characteristics of DXR and share spatial structures, which are very similar to that of Escherichia coli DXR. Phylogenetic and DNA gel blot analyses suggested that a duplication of the DXR gene has occurred in the rubber tree. Semi-quantitative RT-PCR analysis showed that the HbDXR genes are differentially regulated in various tissues of the rubber tree. The HbDXR2 was more highly expressed in clone RRIM 600 than in the wild type, and this is consistent with higher rubber content of this clone. While 2-chloroethane phosphonic acid (ethephon) significantly increased latex yield, it only transiently induced the HbDXR2 gene. The expression of HbDXR2 in the latex suggests its important role in isoprenoid biosynthesis by substrate molecules, indicating that the MEP pathway may have some indirect roles in the biosynthesis of rubber.

Isolation and characterization of two distinct classes of DXS genes in Hevea brasiliensis.

Two cDNAs encoding two distinct classes of DXSs were cloned from leaves (HbDXS1) and latex (HbDXS2) of Hevea brasiliensis by RT-PCR based methods. HbDXS1 encodes a protein of 720 amino acids, with a high homology to the class I of plant DXS proteins, and HbDXS2 encodes a protein predicted to contain 711 amino acids and with a high homology to the plant DXS class II proteins. Several important motifs and amino acid positions characteristic of DXS proteins are strictly conserved in both new HbDXS proteins. The two HbDXS genes were differentially expressed in various tissues of H. brasiliensis. The transcriptional levels of HbDXS2 were similar in both a high-yielding rubber clone (RRIM 600) and the wild type. Ethephon increased the latex yield and caused a transient increase of expression of the HbDXS2 gene. The expression of HbDXS2 in latex indicates that it may have a primary function in carotenoid biosynthesis rather than for natural rubber.