Efficient, green, and rapid strategy for separating actinomycin D and X2 using supercritical fluid chromatography.

Actinomycin D has long been used as a first-line antitumor drug in clinical practice. Actinomycin X2, a new drug lead, is often isolated along with actinomycin D. The minor differences between the two actinomycin analogs pose a daunting challenge in separation. In this study, supercritical fluid chromatography (SFC) was successfully utilized for the purification of actinomycin X2 and actinomycin D from a marine derived Streptomyces sp. DQS-5. After one-step SFC purification, the purities of these two compounds were determined to be 97.3 % and 97.8 %, respectively. This method provides a green alternative for the separation of these pharmacologically important actinomycin antibiotics. This study also demonstrated the development of a simple and rapid method for the separation of natural products based on SFC.

Steroidal Glycosides from Allium tuberosum Seeds and Their Roles in Promoting Testosterone Production of Rat Leydig Cells.

A systematic phytochemical study on the components in the seeds of Allium tuberosum was performed, leading to the isolation of 27 steroidal glycosides (SGs 1-27). The structures of SGs were identified mainly by nuclear magnetic resonance and mass spectrometries as well as the necessary chemical evidence. In the SGs, 1-10 and 22-26 are new steroidal saponin analogues. An in vitro bioassay indicates that 1, 2, 7, 8, 10, 13-15, 20, 23, and 26 display promotional roles in testosterone production of rat Leydig cells with the EC50 values of 1.0 to 4.5 µM, respectively.

Optimization of cellulolytic enzyme components through engineering Trichoderma reesei and on-site fermentation using the soluble inducer for cellulosic ethanol production from corn stover.

BACKGROUND: Cellulolytic enzymes produced by Trichoderma reesei are widely studied for biomass bioconversion, and enzymatic components vary depending on different inducers. In our previous studies, a mixture of glucose and disaccharide (MGD) was developed and used to induce cellulase production. However, the enzymatic profile induced by MGD is still not defined, and further optimization of the enzyme cocktail is also required for efficient ethanol production from lignocellulosic biomass. RESULTS: In this study, cellulolytic enzymes produced by T. reesei Rut C30 using MGD and alkali-pretreated corn stover (APCS) as inducer were compared. Cellular secretome in response to each inducer was analyzed, which revealed a similar enzyme profile. However, significant difference in the content of cellulases and xylanase was detected. Although MGD induction enhanced ß-glucosidase production, its activity was still not sufficient for biomass hydrolysis. To overcome such a disadvantage, aabgl1 encoding ß-glucosidase in Aspergillus aculeatus was heterologously expressed in T. reesei Rut C30 under the control of the pdc1 promoter. The recombinant T. reesei PB-3 strain showed an improved ß-glucosidase activity of 310 CBU/mL in the fed-batch fermentation, 71-folds higher than that produced by the parent strain. Meanwhile, cellulase activity of 50 FPU/mL was detected. Subsequently, the crude enzyme was applied for hydrolyzing corn stover with a solid loading of 20% through separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation, respectively, for ethanol production. Better performance was observed in the SHF process, through which a total of 119.9 g/L glucose was released within 12 h for concomitant ethanol production of 54.2 g/L. CONCLUSIONS: The similar profile of cellulolytic enzymes was detected under the induction of MGD and APCS, but higher amount of cellulases was present in the crude enzyme induced by MGD. However, ß-glucosidase activity induced by MGD was not sufficient for hydrolyzing lignocellulosic biomass. High titers of cellulases and ß-glucosidase were achieved simultaneously by heterologous expression of aabgl1 in T. reesei and fed-batch fermentation through feeding MGD. We demonstrated that on-site cellulase production by T. reesei PB-3 has a potential for efficient biomass saccharification and ethanol production from lignocellulosic biomass.

MACRO: a combined microchip-PCR and microarray system for high-throughput monitoring of genetically modified organisms.

The monitoring of genetically modified organisms (GMOs) is a primary step of GMO regulation. However, there is presently a lack of effective and high-throughput methodologies for specifically and sensitively monitoring most of the commercialized GMOs. Herein, we developed a multiplex amplification on a chip with readout on an oligo microarray (MACRO) system specifically for convenient GMO monitoring. This system is composed of a microchip for multiplex amplification and an oligo microarray for the readout of multiple amplicons, containing a total of 91 targets (18 universal elements, 20 exogenous genes, 45 events, and 8 endogenous reference genes) that covers 97.1% of all GM events that have been commercialized up to 2012. We demonstrate that the specificity of MACRO is ~100%, with a limit of detection (LOD) that is suitable for real-world applications. Moreover, the results obtained of simulated complex samples and blind samples with MACRO were 100% consistent with expectations and the results of independently performed real-time PCRs, respectively. Thus, we believe MACRO is the first system that can be applied for effectively monitoring the majority of the commercialized GMOs in a single test.

Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08.

A monofunctional catalase from Serratia marcescens SYBC08 produced by liquid state fermentation in 7 liter fermenter was isolated and purified by ammonium sulfate precipitation (ASP), ion exchange chromatography (IEC), and gel filtration (GF) and characterized. Its sequence was analyzed by LC-MS/MS technique and gene cloning. The highest catalase production (20,289 U · ml(-1)) was achieved after incubation for 40 h. The purified catalase had an estimated molecular mass of 230 kDa, consisting of four identical subunits of 58 kDa. High specific activity of the catalase (199,584 U · mg(-1) protein) was 3.44 times higher than that of Halomonas sp. Sk1 catalase (57,900 U · mg(-1) protein). The enzyme without peroxidase activity was found to be an atypical electronic spectrum of monofunctional catalase. The apparent K(m) and V(max) were 78 mM and 188, 212 per µM H(2) O(2) µM heme(-1) s(-1), respectivly. The enzyme displayed a broad pH activity range (pH 5.0-11.0), with optimal pH range of 7.0-9.0: It was most active at 20 °C and had 78% activity at 0 °C. Its thermo stability was slightly higher compared to that of commercial catalase from bovine liver. LC-MS/MS analysis confirmed that the deduced amino acid sequence of cloning gene was the catalase sequence from Serratia marcescens SYBC08. The sequence was compared with that of 23 related catalases. Although most of active site residues, NADPH-binding residues, proximal residues of the heme, distal residues of the heme and residues interacting with a water molecule in the enzyme were well conserved in 23 related catalases, weakly conserved residues were found. Its sequence was closely related with that of catalases from pathogenic bacterium in the family Enterobacteriaceae. This result imply that the enzyme with high specific activity plays a significant role in preventing those microorganisms of the family Enterobacteriaceae against hydrogen peroxide resulted in cellular damage. Calalase yield by Serratia marcescens SYBC08 has potential industrial application in scavenging hydrogen peroxide.

From Arabidopsis to rice: pathways in pollen development.

The control of male fertility is of vital importance for crop breeding, hybrid generation, and the control of pollen release. Recent development in the analysis of Arabidopsis male sterile mutants has meant that there is a greater understanding of the gene regulatory networks controlling maternal development of the anther and the resultant sporophytes. With the advent of the genome sequence and tools to allow the analysis of gene function, this knowledge base is now extending into the monocot crop rice. This has shown high levels of similarity between the networks of pollen development in Arabidopsis and rice, which will serve as valuable tools to understand and manipulate this developmental pathway further in plants.

Isolation and identification of a new hypocrellin A-producing strain Shiraia sp. SUPER-H168.

A new hypocrellin A-producing strain, Shiraia sp. SUPER-H168, was isolated from tissues of bamboo, Brachystachyum densiflorum. The morphology of this strain was characterized with a light microscope and a scanning electronic microscope. The mycelia, conidia, pycnidia of fungus were observed. Small pycnidia (10-20 microm in length) full of small conidia appeared on the mycelia, which were first reported in this study. The 18S rDNA region of this strain was amplified and sequenced. Then a neighbor-joining tree of 18S rDNA was constructed. According to the result of analysis, the strain SUPER-H168 was proved to belong to the genus Shiraia. Hypocrellin A was produced by solid-state fermentation, extracted by acetone, isolated by preparative RP-HPLC, and identified by RP-HPLC, ESI-MS and ultraviolet-visible absorbing scanning with diode array detection. The HA production could reach 2.02 mg/g dry solid substrate.

RETARDED PALEA1 controls palea development and floral zygomorphy in rice.

Poaceae, one of the largest flowering plant families in angiosperms, evolved distinct inflorescence and flower morphology diverging from eudicots and other monocots. However, the mechanism underlying the specification of flower morphology in grasses remains unclear. Here we show that floral zygomorphy along the lemma-palea axis in rice (Oryza sativa) is partially or indirectly determined by the CYCLOIDEA (CYC)-like homolog RETARDED PALEA1 (REP1), which regulates palea identity and development. The REP1 gene is only expressed in palea primordium during early flower development, but during later floral stages is radially dispersed in stamens and the vascular bundles of the lemma and palea. The development of palea is significantly retarded in the rep1 mutant and its palea has five vascular bundles, which is similar to the vascular pattern of the wild-type lemma. Furthermore, ectopic expression of REP1 caused the asymmetrical overdifferentiation of the palea cells, altering their floral asymmetry. This work therefore extends the function of the TCP gene family members in defining the diversification of floral morphology in grasses and suggests that a common conserved mechanism controlling floral zygomorphy by CYC-like genes exists in both eudicots and the grasses.

Tapetum degeneration retardation is critical for aliphatic metabolism and gene regulation during rice pollen development.

As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR (Tapetum Degeneration Retardation) gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC-MS (gas chromatography-mass spectrometry) testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C35 in tdr. Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes (236 in total-154 up-regulated and 82 down-regulated) exhibited statistically significant expressional differences between wild-type and tdr. In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development.

[Introduction of Plasmodium falciparum C-terminal region of the merozoite surface protein gene into the chloroplast of tobacco].

OBJECTIVE: To construct chloroplast expression vector, and introduce the C-terminal region of the merozoite surface protein 1 gene (msp1-42) of Plasmodium falciparum 3D7 strain into the chloroplast genome of tobacco for expression of the recombinant protein MSP1-42. METHODS: Forward and reverse primers, adjusted to tobacco chloroplast codon preferences, were used for generation of msp1-42 gene from pBluntmsp plasmid which contains msp1-42 gene. A chloroplast expression vector LRrrmsp was constructed and bombarded into leaves of tobacco by a biolistic He particle delivery system. Media containing 500 mg/L spectinomycin were used for selection of spectinomycin resistant plant. PCR was carried out to check the introduction of the msp1-42 and aadA genes into the chloroplast genome. The transgenic plants with msp1-42 and aadA gene insertion were cut and cultured on the generation MS media containing 500 mg/L spectinomycin for at least 3 turns, and multiple PCR were applied to analyse their homogenization. RESULTS: A chloroplast expression vector LRrrmsp was constructed and confirmed with PCR and enzyme digestion analysis. Six transformants were obtained with a transformation rate 0.6/gun. The msp1-42 and aadA genes were amplified from spectinomycin resistant plants by PCR detection. Wild type chloroplast gene was detected by multiple-PCR analysis. CONCLUSION: A chloroplast expression vector containing msp1-42 gene was constructed. The msp1-42 gene was introduced into chloroplast genome of tobacco and heterogeneous transgenic tobacco was obtained.

Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum.

This report provides the complete nucleotide sequences of the full-length cDNA encoding squalene synthase (SQS) and its genomic DNA sequence from a triterpene-producing fungus, Ganoderma lucidum. The cDNA of the squalene synthase (SQS) (GenBank Accession Number: DQ494674) was found to contain an open reading frame (ORF) of 1,404 bp encoding a 468-amino-acid polypeptide, whereas the SQS genomic DNA sequence (GenBank Accession Number: DQ494675) consisted of 1,984 bp and contained four exons and three introns. Only one gene copy was present in the G lucidum genome. The deduced amino acid sequence of Ganoderma lucidum squalene synthase (Gl-SQS) exhibited a high homology with other fungal squalene synthase genes and contained six conserved domains. A phylogenetic analysis revealed that G. lucidum SQS belonged to the fungi SQS group, and was more closely related to the SQS of U. maydis than to those of other fungi. A gene expression analysis showed that the expression level was relatively low in mycelia incubated for 12 days, increased after 14 to 20 days of incubation, and reached a relatively high level in the mushroom primordia. Functional complementation of Gl-SQS in a SQS-deficient strain of Saccharomyces cerevisiae confirmed that the cloned cDNA encoded a squalene synthase.

[Molecular cloning and analysis of the 3' terminal sequence of duck hepatitis virus genome].

The authentic 3' terminal sequences of genomes of duck hepatitis virus (DHV) serotype I strain C80 and serotype I variant strain E63 were obtained by using 3' RACE and RT-PCR techniques. The analysis showed that 3' terminal sequences in the genomes of the two DHV strains include the 3D region of 1359 nucleotides (nt), the terminator codon TGA, and 3'untranslated region (UTR) of 314 nt. The poly (A) tails of C80 and E63 are 18 nt and 19 nt in length respectively. The deduced 3D proteins of 453 amino acids of both DHV strains contain the motifs KDELR, DxxxxD, GxxCSGxxxTxxxNS, YGDD, and FLKR characteristic for RNA polymerase of picornaviruses, which confirms DHV serotype I to be a member of Picornaviridae . The amino acid sequence identities among 3D protein of the two DHV strains with representatives of nine other picornavirus genera range from 16% to 37%, which are within the value ranges (18%-60%) of 3D amino acid sequence identities obtained from inter-genera comparisons. In addition, DHV serotype I possesses the longest 3'UTR in the family Picornaviridae. Phylogenetic analysis of 3D proteins indicated that DHV serotype I may belong to a separated genus of the family Picornaviridae.

The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development.

In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.

[Identification of the rice (Oryza sativa L.) mutant msp1-4 and expression analysis of its UDT1 and GAMYB genes].

A rice male-sterile mutant msp1-4 (MULTIPLE SPOROCYTE) with japonica cultivar '9522' background, was obtained in M(3) population treated with (60)Co gamma-ray. Results of genetic analysis indicated that the male-sterile phenotype was controlled by a single recessive locus. To map this locus, an F(2) population was constructed from the cross between the msp1-4 (japonica) and 'LongTeFu B' (indica). This locus was mapped between the two InDel markers, WY-4 and WY-8, with physical distance of 247 kb. A deletion with 10 base pairs between 758 bp and 767 bp in MSP1 open reading frame was confirmed by sequence analysis, which led to pre-termination of MSP1 translation. Phenotype analysis of msp1-4 indicated that it was similar to the msp1 mutant. To insight the expression change of rice anther developmental genes in this mutant, semi-quantitative RT-PCR analysis was carried out. The results showed that the expression level of rice UDT1 and GAMYB were reduced in msp1-4, implying that UDT1 and GAMYB are possibly the downstream genes of MSP1 gene in rice pollen development.

[Rapid identification of Riemerella anatipestifer on the basis of specific PCR amplifying 16S rDNA].

Riemerella anatipestifer (RA) infection is the main disease causing severe losses in duck production. Because RA is characterized more by the absence than by the presence of specific phenotypic properties and different scholar had the different results of biochemical detection, it can't always be identified quickly and correctly only by the phenotypic properties or biochemical characteristics. The research object was to develop a species specific PCR method for RA detection. Because of the conserved structure of rRNA and appropriate size of 16S rRNA, a multiple alignment of 16S rDNA (gene coding 16S rRNA) was processed among RA, Escherichia coli, Pasteurella multocida, Salmonella enteritidis and Salmonella gallinarum, which are the main bacteria causing duck diseases. A pair of species specific primers named 190f and 843r were selected from the variable regions of 16S rDNA depending on the result of multiple alignment. Using BLAST on NCBI website for a sequence similarity search, the results showed that this pair of primers had very high specificity, except for having a lower sequence similarity with some species of Flavobacterium and Chryseobacterium. A PCR assay was performed and the template was extracted using the bacteria genomic DNA extraction kit and boiling method respectively. A chelate resin named Chelex 100 was used in the boiling method at the same time. Under the annealing temperature of 60 degrees C, all the 26 RA strains, including 19 representative strains of serotypes 1 to approximately 19 and 7 domestic isolated strains, showed the same 654bp fragment after PCR, while there was no amplification with isolates of other bacterial species. Also a series of sensitivity experiments were performed and proved that the detection limit of this method was 50pg genomic DNA, 1.5 x 10(6) CFU/mL and 15 CFU/mL, when the template was prepared with genomic DNA extraction kit, only boiling method and boiling method with Chelex 100 respectively. 12 clinical cases which were probably infected with RA were chosen to identify the accuracy and sensitivity of this PCR method. Several other conventional detecting methods including bacteria isolating, differentiating culture, biochemical experiments and serotyping were used at the same time. The templates of PCR were extracted from brains or livers by boiling method with Chelex 100. Finally, 3 cases were identified as RA infection by the conventional methods and 4 by the PCR method, which proved the good accuracy and sensitivity of the PCR method. Thus, this PCR assay provides a rapid and accurate method for identification of Riemerella anatipestifer. It will help to make the final decision in clinical diagnose or species identification, especially when a new serotype or sub-serotype of RA comes up.

[Genetic analysis and mapping of the rice leafy-hull mutant Oslh].

Oslh (lh=leafy hull), in the japonica cultivar 9522 background, a mutant of Oryza sativa L. spp. japonica cv. 9522 identified from an M(2) population, was mutagenized by irradiation with (60)Co gamma-ray. The Oslh mutant plants flowered about 15 days later than the wild-type plants (Fig.1e). The paleas, lemmas and lodicules of the flowers of Oslh mutant were transformed into leaf-like structures (Fig.1b, d). Genetic analysis of the F(2) progeny from a cross between the Oslh mutant and wild-type japonica cv. 9522 revealed that the Oslh mutant arouse from a single recessive nuclear gene mutation of the cv. 9522. To map the Oslh locus, an F(2) population generated by crossing between Oslh (japonica) mutant and Guangluai4 (indica) was analyzed. The Oslh locus was mapped to the long arm of rice chromosome 3, between a SSR marker RM5475 and an InDel marker GY305, 2.9 and 1.5 cM away from these two markers respectively (Fig.4). These results are useful for further cloning and functional analysis of the OsLH gene.

[Cloning of CTB-PROIN fusion gene and its expression in Escherichia coli].

A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.

Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes.

Here, we report our effort in generating an ORFeome collection for the Arabidopsis transcription factor (TF) genes. In total, ORFeome clones representing 1,282 Arabidopsis TF genes have been obtained in the Gateway high throughput cloning pENTR vector, including 411 genes whose annotation lack cDNA support. All the ORFeome inserts have also been mobilized into a yeast expression destination vector, with an estimated 85% rate of expressing the respective proteins. Sequence analysis of these clones revealed that 34 of them did not match with either the reported cDNAs or current predicted open-reading-frame sequences. Among those, novel alternative splicing of TF gene transcripts is responsible for the observed differences in at least five genes. However, those alternative splicing events do not appear to be differentially regulated among distinct Arabidopsis tissues examined. Lastly, expression of those TF genes in 17 distinct Arabidopsis organ types and the cultured cells was profiled using a 70-mer oligo microarray.

Purification of the recombinant hepatitis B virus core antigen (rHBcAg) produced in the yeast Saccharomyces cerevisiae and comparative observation of its particles by transmission electron microscopy (TEM) and atomic force microscopy (AFM).

Hepatitis B virus core antigen (HBcAg) gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg or core particles) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, Sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. It has been observed that HBcAg was synthesized in yeast cells as a particle consisting of polypeptides with a molecular weight of 21.5 kDa (p21.5). Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (rHBcAg particles) with HBcAg antigenicity mainly located at the densities of 1.27 and 1.40 g ml(-1), respectively. Observation and analysis of the purified rHBcAg products by TEM indicated that rHBcAg peptides could mainly self-assemble into two size classes of core particles. The larger particles were approximately 30.1 nm and the smaller were approximately 21.5 nm in mean diameter. Further observation and analysis of the same rHBcAg (core) particles by AFM also indicated that rHBcAg (core) particles were similar to the native HBcAg (core) particles from infected human hepatocytes and mainly composed of two size classes of partides core. The larger particles were approximately 31.3 nm and the smaller were approximately 22.5 nm in mean diameter which was similar to the results obtained by TEM. All results from both TEM and AFM suggested that core particles (capsids) produced in S. cerevisiae possessed dimorphism.

Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs.

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.