Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml^?1 of bacterial cells and spores, respectively. IPCR was found to be 2–3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.     

Isolation and in silico analysis of Fe-superoxide dismutase in the cyanobacterium Nostoc commune

Cyanobacteria are known to endure various stress conditions due to the inbuilt potential for oxidative stress alleviation owing to the presence of an array of antioxidants. The present study shows that Antarctic cyanobacterium Nostoc commune possesses two antioxidative enzymes viz., superoxide dismutase (SOD) and catalase that jointly cope with environmental stresses prevailing at its natural habitat. Native-PAGE analysis illustrates the presence of a single prominent isoform recognized as Fe-SOD and three distinct isoforms of catalase. The protein sequence of Fe-SOD in N. commune retrieved from NCBI protein sequence database was used for in silico analysis. 3D structure of N. commune was predicted by comparative modeling using MODELLER 9v11. Further, this model was validated for its quality by Ramachandran plot, ERRAT, Verify 3D and ProSA-web which revealed good structure quality of the model. Multiple sequence alignment showed high conservation in N and C-terminal domain regions along with all metal binding positions in Fe-SOD which were also found to be highly conserved in all 28 cyanobacterial species under study, including N. commune. In silico prediction of isoelectric point and molecular weight of Fe-SOD was found to be 5.48 and 22,342.98 Da respectively. The phylogenetic tree revealed that among 28 cyanobacterial species, Fe-SOD in N. commune was the closest evolutionary homolog of Fe-SOD in Nostoc punctiforme as evident by strong bootstrap value. Thus, N. commune may serve as a good biological model for studies related to survival of life under extreme conditions prevailing at the Antarctic region. Moreover cyanobacteria may be exploited for biochemical and biotechnological applications of enzymatic antioxidants.     

A highly sensitive immuno-PCR assay for detection of H5N1 avian influenza virus

With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID₅₀ ml⁻¹~10⁻⁴ EID₅₀ ml⁻¹,which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 μl of AIV H5N1 with 10⁻⁴ EID₅₀ ml⁻¹.To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT–PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT–PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal–tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.     

Nested Inverse Polymerase Chain Reactions: An Effective Method for Cloning of Full-Length Sequences of Chalcone Synthase

Chalcone synthase is the key enzyme in biosynthesis of flavonoids, which play roles in pigmentation of flowers and protection against ultraviolet and pathogens. Inverse polymerase chain reaction (IPCR) is a method for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. In this study, IPCR united with nested PCR was successfully applied in cloning full-length sequences of three Phalaenopsis chalcone synthase genes (phchs3, phchs4, and phchs5, respectively). Firstly, routine PCR with homologous primers were performed, and gene fragments of phchs3 (1 kb), phchs4 (1.2 kb), and phchs5 (800 bp) were obtained and then sequenced. Then, inverse PCR were carried out for cloning full-length sequence of each gene. Because products were not unique in single round inverse PCR, nested PCR were performed, and the specificity was much enhanced. At last, full-length sequences of 2,499 bp for phchs3, 2,502 bp for phchs4, and 1,855 bp for phchs5 were obtained. This study proved that IPCR could be more efficient if being united with nested PCR.     

Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli

Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78^T was created in λ zap express™ and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 825 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastrointestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) α subunit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identity in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).     

The superoxide dismutase-encoding gene of the obligately anaerobic bacterium Bacteroides gingivalis

The gene (sod) encoding the superoxide dismutase (SOD) of the obligately anaerobic bacterium Bacteroides gingivalis was cloned. The amino acid (aa) sequence of the SOD, deduced from the nucleotide sequence of the sod gene, basically resembled that of known Fe-SODs. However, the aa sequence of the B. gingivalis SOD was found to be intermediate between those of Fe-SOD and Mn-SOD in a limited region around the putative second ligand, where major differences between the aa sequences of Fe-SOD and Mn-SOD are known to exist.     

Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus

A gene (empV) encoding the extracellular metalloprotease of Vibrio vulnificus CKM-1 has been cloned and sequenced. When the empV gene was expressed in minicells, a unique peptide of approx. 46 kDa was identified. Protease activity staining experiments also indicated a similar M_r for the protease. The empV gene product (EmpV) is secreted into the periplasm of Escherichia coli, but not out of it. The crude enzyme prepared from the periplasmic fraction of recombinant E. coli was inhibited by a metalloprotease inhibitor and Zn²⁺ is essential for its protease activity. Nucleotide sequence analysis predicted a single open reading frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long `pro' sequence consisting of 172 aa. The N-terminal 20 aa sequence for the elastolytic protease (EepV), purified from the culture supernatant of V. vulnificus ATCC 29307, completely identified the beginning of the predicted mature protein within the deduced aa sequence except for 1 aa residue difference. The estimated pI and molecular weight of the predicted mature protein were 5.86 and 44.3 kDa, respectively, which are nearly identical to those of V. vulnificus L-180 extracellular neutral metalloprotease (EnmV) and of strain ATCC 29307 EepV. The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of the minicells and by protease activity staining. The deduced aa sequence of EmpV showed high homology to V. anguillarum metalloprotease (EmpA), V. cholerae HA/protease (HprC), and V. proteolyticus neutral protease (NprP), particularly with respect to active-site residues, zinc-binding residues, and cysteine residues.     

Isolation and sequence analysis of Clpgl,a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum

Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race β and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed an ORF encoding a 363-amino-acid (aa) polypeptide begining with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5′ non-coding region. This genomic clone was thereafter designated Clpgl. Southern analysis, performed with a Clpgl-specific probe, showed that this gene is present as a single copy in the Cl genome     

Expression and characterization of an iron-containing superoxide dismutase from Burkholderia pseudomallei

A superoxide dismutase (SOD) gene from Burkholderia pseudomallei, the causative agent of melioidosis, was cloned and expressed in Escherichia coli, and its product was functionally and physically characterized. The gene has an open-reading frame of 579 bp. The deduced amino acid sequence has 192 residues with a calculated molecular mass of ～22 kDa. Sequence comparison with other bacterial SODs showed that the protein contains typical metal-binding motifs and other Fe-SOD-conserved residues. The sequence has substantial similarity with other bacterial Fe-SOD sequences. The enzymatic activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide or potassium cyanide, attributes that indeed are characteristic of typical bacterial Fe-SODs. Western blotting with antiserum against the recombinant Fe-SOD revealed that it is expressed in B. pseudomallei. Transformed E. coli that expressed the Fe-SOD had significantly increased SOD activity and was highly tolerant to paraquat-mediated replication inhibition, compared to transformed cells carrying an empty vector. Our results provide a basis for further biochemical characterization of the enzyme and elucidation of its role in the pathogenesis of B. pseudomallei.     

Classification of catechol 1,2-dioxygenase family: sequence analysis of a gene for the catechol 1,2-dioxygenase showing high specificity for methylcatechols from Gram+ aniline-assimilating Rhodococcus erythropolis AN-13

Gram⁺ aniline-assimilating Rhodococcus erythropolis AN-13 (AN-13) produces catechol 1,2-dioxygenase (C12O) showing high enzymatic activities for 3- and 4-methylcatechols [Aoki et al. (1984) Agric. Biol. Chem. 48, 2087–2095]. A 3.0 kb Sau3AI fragment carrying a gene encoding C12O (catA) was cloned by selection of transformants showing C12O activity from a gene library of AN-13. Furthermore, we specified a 1.6 kb SalI fragment containing catA from the Sau3AI fragment by subcloning. Sequence analysis revealed that the 1.6 kb SalI fragment carried a 855 bp open reading frame (ORF) encoding the entire AN-13 catA, preceded by a potential ribosome binding site (RBS). From comparison of the deduced amino acid (aa) sequence of C12O from AN-13 with other C12O reported previously, it was found that the AN-13 enzyme shares 56.0% aa sequence identity with C12O from Arthrobacter sp. mA3 (mA3) [Eck and Belter (1991) Gene 123, 87–92] compared with less than 36.4% aa sequence identities with others. In conclusion, we classified all C12O including the AN-13 enzyme into three subfamilies on the basis of similarity of aa sequences, numbers of aa residues, and substrate specificity.     

Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10⁴ times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.     

Molecular cloning of the complete 11S seed storage protein gene of Coffea arabica and promoter analysis in transgenic tobacco plants

In this paper, we present the complete nucleotide sequence of the csp1 gene from Coffea arabica coding for the 11S-globulin seed storage protein. To investigate the sequences responsible for the regulated expression of this seed-specific coffee storage protein gene, about 1 kb of the 5'-upstream region from the csp1 gene was isolated using inverse polymerase chain reaction (IPCR) and then sequenced. Several DNA boxes were found in this coffee sequence that had similarity to those previously identified as being essential for grain (endosperm) specific expression in other plants. To study the ability of this sequence to direct grain-specific expression, the whole fragment, as well as a series of 5' deletions, was fused to the reporter gene β-glucuronidase (uidA) and analysed in transgenic Nicotiana tabacum plants. GUS measurements showed that all the deletions of the csp1 promoter directed the expression of the reporter gene in tobacco grain but not in the other tissues examined. GUS activities also revealed that the csp1 promoter constructs function as very strong promoters by comparison to the strength of the cauliflower mosaic virus (CaMV) 35S promoter. Therefore, this 11S promoter could represent a useful tool to change the expression of targeted genes in the grain of transgenic coffee plants.     

An ultrasensitive gold nanoparticles improved real-time immuno-PCR assay for detecting diethyl phthalate in foodstuff samples

A specific polyclonal antibody targeting diethyl phthalate (DEP) with the higher antibody titer at 1:120,000 has been obtained, and an ultrasensitive and high-throughput direct competitive gold nanoparticles improved real-time immuno-PCR (GNP–rt–IPCR) technique has been developed for detecting DEP in foodstuff samples. Under optimal conditions, a rather low linearity is achieved within a range of 4 pg L⁻¹ to 40 ng L⁻¹, and the limit of detection (LOD) is 1.06 pg L⁻¹. Otherwise, the GNP–rt–IPCR technique is highly selective, with low cross-reactivity values for DEP analogs (<5%). Finally, the concentrations of DEP in foodstuff samples by the GNP–rt–IPCR method range from 0.48 to 41.88 μg kg⁻¹. Satisfactory recoveries (88.39–112.79%) and coefficient of variation values (8.38–12.77%) are obtained. The consistency between the results obtained from GNP–rt–IPCR and gas chromatography–mass spectrometry (GC–MS) is 98.3%, which further proves that GNP–rt–IPCR is an accurate, reliable, rapid, ultrasensitive, and high-throughput method for batch determination of trace amounts of DEP in foodstuff samples.     

Molecular characterization and bioactivity of a CXCL13 chemokine in large yellow croaker Pseudosciaena crocea

A CXCL13-like chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCXCL13). The full-length cDNA of LycCXCL13 is 796 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 10.7 kDa. The deduced LycCXCL13 contains a 24-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines (C²⁵, C²⁷, C⁵² and C⁶⁸). It shares 35, 36 and 39% aa sequence identities to green puffer CXCL13-like, Atlantic salmon CXCL13 and Japanese flounder CXCL13 chemokines, and 24–29% identities to CXCL13 chemokines in mammals, respectively. Phylogenetic analysis showed that LycCXCL13 is more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups. LycCXCL13 gene was constitutively expressed in all tissues examined, except for intestine. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL13 gene expression was significantly up-regulated in spleen, head kidney, heart and gills at 24 h post-injection. Real-time PCR results showed that LycCXCL13 gene expression reached peak level in spleen and head kidney at 12 h after induction by poly(I:C), while its expression increased to the highest level in head kidney at 24 h or in spleen at 48 h by bacterial vaccine. Recombinant LycCXCL13 protein produced in E. coli BL21 exhibited obvious chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggest that LycCXCL13 may be involved in inflammatory responses as well as homeostatic processes in large yellow croaker.     

Sequence of a gene encoding a putative primary sigma factor from Borrelia burgdorferi strain B31

Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa). Comparison with Escherichia coli σ⁷⁰ and Bacillus subtilis σ⁴³ showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding.     

Modified pPIC9K vector-mediated expression of a family 11 xylanase gene, Aoxyn11A, from Aspergillus oryzae in Pichia pastoris

A full-length cDNA sequence of Aoxyn11A, a mesophilic xylanase-encoding gene from Aspergillus oryzae, was obtained from total RNA, using 3′ and 5′ rapid amplification of cDNA ends methods. The cDNA sequence is 1,086 base pairs in length, containing 5′-untranslated and 3′-untranslated regions and an open reading frame encoding a 20 amino acid (aa) signal peptide, a 24 aa propeptide and a 188 aa mature peptide (designated AoXyn11A). Multiple alignments verified that AoXyn11A belongs to glycoside hydrolase family 11. Its three-dimensional structure was predicted by multiple templates–based homology modeling. In addition, an AoXyn11A-encoding cDNA gene was extracellularly expressed in Pichia pastoris GS115, mediated by the modified pPIC9K vector. One P. pastoris transformant, numbered as GSAorX4-3 and having the highest recombinant AoXyn11A (reAoXyn11A) activity of 98.0 U/ml, was chosen. The reAoXyn11A showed maximum activity at pH 5.5 and 50 °C. It was highly stable at a pH range of 4.0–8.0 and at 40 °C. Its activity was not significantly affected by metal ions that were tested or EDTA, but was strongly inhibited by Mn²⁺ and Ag⁺. The K _m and V _max of the reAoXyn11A were 1.85 mg/ml and 3,018 U/mg, respectively.     

Direct colony PCR for rapid identification of varied microalgae from freshwater environment

Molecular identification of microalgal species is vital and involves sequencing of specific markers present in the genome, which are unique to a genus. PCR is a vital tool for identification of microalgae; the preparation of template DNA for PCR by traditional methods requires large amount of algal cells and a time-consuming process. One simple way to reduce these complications is to use the microalgal colonies directly for amplification of required DNA fragments from the genome. In this study, a simple cell-disrupting method, using autoclaved glass powder, has been used for direct colony PCR of microalgae. Four morphologically different microalgal strains were chosen from freshwater samples, and the PCR amplification reaction was evaluated with three different molecular markers (rbcL, internal transcribed spacer 2, and 18S rDNA). PCR amplification was optimized with less number of cells (0.04?×?10⁵), minimal quantity of glass powder (0.5 mg), and in the presence of Milli-Q water for internal transcribed spacer marker. The isolated strains were identified as Desmodesmus sp. JQ782747, Coelastrum proboscideum JQ898144, Chlorella sorokiniana JQ898145, and Scenedesmus sp. JQ782746 based on sequence similarity. This direct microalgal colony PCR proves to be a simple and rapid method for detection of varied microalgal species.