Corrigendum: Development of sandwich dot-ELISA for specific detection of Ochratoxin A and its application on to contaminated cereal grains originating from India.

[This corrects the article DOI: 10.3389/fmicb.2015.00511.].

Development of Hybrid IgG-Aptamer Sandwich Immunoassay Platform for Aflatoxin B1 Detection and Its Evaluation Onto Various Field Samples.

The present study was aimed to develop a novel antibody-aptamer based hybrid detection strategy for specific and sensitive detection of aflatoxin B1 (AFB1) from contaminated food grains. The study comprises generation of ssDNA aptamers and anti-AFB1 IgG against AFB1 toxin. The generated bio-probes (aptamers and antibodies) were further characterized for their specificity and sensitivity using indirect ELISA. The generated aptamers namely AFB1a and AFB1b showed prominent reactivity and selectivity against AFB1 toxin. These aptamers were further characterized for their secondary structures and dG values were determined as -4.6 and -2.75 Kcal/mol, respectively. The detection limit (LOD) of AFB1a and anti-AFB1 IgG was determined as 5 and 10 ng/mL, respectively. The characterized aptamers and antibodies against AFB1 were used to develop the sandwich immunoassay. Anti AFB1 IgG was used as a capturing antibody whereas anti-AFB1a aptamer was used as its revealing partner in the assay. The limit of detection (LOD) of the immunoassay was determined to be 5 ng/mL of AFB1 standard toxin and showed no cross-reactivity with closely related mycotoxins. To assess the reliability of the developed method, several field samples contaminated with aflatoxin B1 was included in the study and results were validated with commercial AFB1-ELISA Kit. Additionally, the spiking studies were also carried out to demonstrate the consistency and dependability of the developed hybrid sandwich immunoassay wherein the toxins recovered were found to be ranging between 73 and 98.80% with the LOD at 5 ng/mL. In conclusion, the developed method may find the better utility in routine food testing laboratories for assessment of AFB1.

Development and evaluation of an IgY based silica matrix immunoassay platform for rapid onsite SEB detection.

The present study involves immunoassay platform development based on a surface functionalized silica matrix for rapid onsite detection of Staphylococcal enterotoxin B (SEB). Silica matrix functionalization as well as the immunoassay parameters was experimentally designed and optimized through response surface methodology (RSM). Silica surface functionalization was carried out with hydrofluoric acid (HF), ammonia, 3-aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The RSM optimized matrix functionalization parameters for HF, ammonia, APTES and GA were determined to be 10%, 40%, 20% and 10% (V/V), respectively. Antibodies for the study were generated against recombinant SEB toxin in rabbit (anti-SEB IgG) and chicken (anti-SEB IgY). Subsequently, antibodies were immobilized on the functionalized silica matrix and were further characterized by SEM and contact angle measurements to elucidate the surface uniformity and degree of hydrophilicity. The immunoassay platform was developed with anti-SEB IgG (capturing agent) and anti-SEB IgY (revealing partner). The limit of detection (LOD) of the developed platform was determined to be 0.005 µg mL-1 and no cross-reactivity with similar toxins was observed. Upon co-evaluation with a standard ELISA kit (Chondrex, Inc) against various field isolates, the platform was found to be on par and reliable. In conclusion, the developed method may find better utility in onsite detection of SEB from resource-poor settings.

Co-circulation and co-infections of all dengue virus serotypes in Hyderabad, India 2014.

The burden of dengue virus infections increased globally during recent years. Though India is considered as dengue hyper-endemic country, limited data are available on disease epidemiology. The present study includes molecular characterization of dengue virus strains occurred in Hyderabad, India, during the year 2014. A total of 120 febrile cases were recruited for this study, which includes only children and 41 were serologically confirmed for dengue positive infections using non-structural (NS1) and/or IgG/IgM ELISA tests. RT-PCR, nucleotide sequencing and evolutionary analyses were carried out to identify the circulating serotypes/genotypes. The data indicated a high percent of severe dengue (63%) in primary infections. Simultaneous circulation of all four serotypes and co-infections were observed for the first time in Hyderabad, India. In total, 15 patients were co-infected with more than one dengue serotype and 12 (80%) of them had severe dengue. One of the striking findings of the present study is the identification of serotype Den-1 as the first report from this region and this strain showed close relatedness to the Thailand 1980 strains but not to any of the strains reported from India until now. Phylogenetically, all four strains of the present study showed close relatedness to the strains, which are reported to be high virulent.

Complete genome characterization and evolutionary analysis of serotype-4 associated with severe dengue.

Dengue virus circulates as four independent serotypes posing a major public health threat around the globe. In the recent years, frequent dengue outbreaks are being reported in many parts of the world including India. Among four serotypes, Den-4 is the least sampled and studied serotype until recent times, but the reported cases with Den-4 infections were mostly known to associate with severe dengue. In the past three decades, only one complete genome sequence of Den-4 has been published from India. Hence there is a deficit in information with reference to this serotype which would be required in deciphering its association with severe dengue. In this study, we have carried out the complete genome characterization of Den-4 virus, isolated from a dengue shock syndrome patient during the 2015 outbreak from Hyderabad, South India. Phylogenetic analysis revealed the circulation of genotype I (lineage C) which showed close relatedness to the reported virulent strains. The data also indicated few unique amino acid substitutions which are known to be important in virus replication and epitope presentation. This is the first report of complete genome characterization of Den-4 from South India, which may assist in shaping the genetic diversity of circulating strains in India.

Molecular phylogeny, pathogenicity and toxigenicity of Fusarium oxysporum f. sp. lycopersici.

The present study aimed at the molecular characterization of pathogenic and non pathogenic F. oxysporum f. sp. lycopersici strains isolated from tomato. The causal agent isolated from symptomatic plants and soil samples was identified based on morphological and molecular analyses. Pathogenicity testing of 69 strains on five susceptible tomato varieties showed 45% of the strains were highly virulent and 30% were moderately virulent. Molecular analysis based on the fingerprints obtained through ISSR indicated the presence of wide genetic diversity among the strains. Phylogenetic analysis based on ITS sequences showed the presence of at least four evolutionary lineages of the pathogen. The clustering of F. oxysporum with non pathogenic isolates and with the members of other formae speciales indicated polyphyletic origin of F. oxysporum f. sp. lycopersici. Further analysis revealed intraspecies variability and nucleotide insertions or deletions in the ITS region among the strains in the study and the observed variations were found to be clade specific. The high genetic diversity in the pathogen population demands for development of effective resistance breeding programs in tomato. Among the pathogenic strains tested, toxigenic strains harbored the Fum1 gene clearly indicating that the strains infecting tomato crops have the potential to produce Fumonisin.

Rapid onsite detection of bacterial spores of biothreat importance by paper-based colorimetric method using erbium-pyrocatechol violet complex.

Dipicolinic acid (DPA) is an important chemical marker for the detection of bacterial spores. In this study, complexes of lanthanide series elements such as erbium, europium, neodymium, and terbium were prepared with pyrocatechol violet and effectively immobilized the pyrocatechol violet (PV)-metal complex on a filter paper using polyvinyl alcohol. These filter paper strips were employed for the onsite detection of bacterial spores. The test filter papers were evaluated quantitatively with different concentrations of DPA and spores of various bacteria. Among the four lanthanide ions, erbium displayed better sensitivity than the other ions. The limit of detection of this test for DPA was 60 µM and 5 × 10(6) spores. The effect of other non-spore-forming bacteria and interfering chemicals on the test strips was also evaluated. The non-spore-forming bacteria did not have considerable effect on the test strip whereas chemicals such as EDTA had significant effects on the test results. The present test is rapid and robust, capable of providing timely results for better judgement to save resources on unnecessary decontamination procedures during false alarms.

Cytotoxic effects of oosporein isolated from endophytic fungus Cochliobolus kusanoi.

In the present study, oosporein, a fungal toxic secondary metabolite known to be a toxic agent causing chronic disorders in animals, was isolated from fungus Cochliobolus kusanoi of Nerium oleander L. Toxic effects of oosporein and the possible mechanisms of cytotoxicity as well as the role of oxidative stress in cytotoxicity to Madin-Darby canine kidney kidney cells and RAW 264.7 splene cells were evaluated in vitro. Also to know the possible in vivo toxic effects of oosporein on kidney and spleen, Balb/C mouse were treated with different concentrations of oosporein ranging from 20 to 200 µM). After 24 h of exposure histopathological observations were made to know the effects of oosporein on target organs. Oosporein induced elevated levels of reactive oxygen species (ROS) generation and high levels of malondialdehyde, loss of mitochondrial membrane potential, induced glutathione hydroxylase (GSH) production was observed in a dose depended manner. Effects oosporein on chromosomal DNA damage was assessed by Comet assay, and increase in DNA damage were observed in both the studied cell lines by increasing the oosporein concentration. Further, oosporein treatment to studied cell lines indicated significant suppression of oxidative stress related gene (Superoxide dismutase1 and Catalase ) expression, and increased levels of mRNA expression in apoptosis or oxidative stress inducing genes HSP70, Caspase3, Caspase6, and Caspase9 as measured by quantitative real time-PCR assay. Histopathological examination of oosporein treated mouse kidney and splenocytes further revealed that, oosporein treated target mouse tissues were significantly damaged with that of untreated sam control mice and these effects were in directly proportional to the the toxin dose. Results of the present study reveals that, ROS is the principle event prompting increased oosporein toxicity in studied in vivio and in vitro animal models. The high previlance of these fungi in temperate climates further warrants the need of safe food grain storage and processing practices to control the toxic effects of oosporein to humans and live stock.

Development of sandwich dot-ELISA for specific detection of Ochratoxin A and its application on to contaminated cereal grains originating from India.

In the present study, generation and characterization of a highly specific monoclonal antibody (mAb) against Ochratoxin A (OTA) was undertaken. The generated mAb was further used to develop a simple, fast, and sensitive sandwich dot-ELISA (s-dot ELISA) method for detection of OTA from contaminated food grain samples. The limit of detection (LOD) of the developed enzyme-linked immunosorbent assay (ELISA) method was determined as 5.0 ng/mL of OTA. Developed method was more specific toward OTA and no cross reactivity was observed with the other tested mycotoxins such as deoxynivalenol, fumonisin B1, or aflatoxin B1. To assess the utility and reliability of the developed method, several field samples of maize, wheat and rice (n = 195) collected from different geographical regions of southern Karnataka region of India were evaluated for the OTA occurrence. Seventy two out of 195 samples (19 maize, 38 wheat, and 15 rice) were found to be contaminated by OTA by s-dot ELISA. The assay results were further co-evaluated with conventional analytical high-performance liquid chromatography (HPLC) method. Results of the s-dot ELISA are in concordance with HPLC except for three samples that were negative for OTA presence by s-dot ELISA but found positive by HPLC. Although positive by HPLC, the amount of OTA in the three samples was found to be lesser than the accepted levels (>5 µg/kg) of OTA presence in cereals. Therefore, in conclusion, the developed s-dot ELISA is a better alternative for routine cereal based food and feed analysis in diagnostic labs to check the presence of OTA over existing conventional culture based, tedious analytical methods.

Analysis of genetic and aflatoxin diversity among Aspergillus flavus isolates collected from sorghum seeds.

Thirty-four Aspergillus flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) quantification of total aflatoxin concentrations by the indirect competitive-ELISA (ic-ELISA) method, and (3) analysis of molecular diversity among the A. flavus isolates using ß-tubulin, ITS, and ISSR markers. Among the isolates studied, 28 were found to be positive for the production of aflatoxins. ITS and ß-tubulin phylogenetic analysis segregated the A. flavus sample population into two major groups or clades with little to no subdivision based on geography. In contrast, ISSR analysis also separated the A. flavus isolates into two main clusters, showing a distance of 0.0-0.5, with one cluster exhibiting a high level of diversity though no geographic or chemotype subdivision could be observed. The majority of sampled A. flavus isolates were highly toxigenic, and also highly diversified in terms of toxin-producing potential in-vitro. Genetic diversity among the sorghum isolates of A. flavus further warrants the development of appropriate farming management practices as well as improved aflatoxin detection measures in India.

Cytotoxic effect of p-Coumaric acid on neuroblastoma, N2a cell via generation of reactive oxygen species leading to dysfunction of mitochondria inducing apoptosis and autophagy.

p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against pathological conditions. In the present study, p-CA was evaluated for its capacity to induce cytotoxic effect to neuroblastoma N2a cells and we report here the possible mechanism of its action. p-CA at a concentration of 150 µmol/L, upon exposure for 72 h, stimulated 81.23 % of cells to apoptosis, as evidenced by flow cytometer studies mediated through elevated levels of ROS (7.5-fold over control). Excess ROS production activated structural injury to mitochondrial membrane, observed as dissipation of its membrane potential and followed by the release of cytochrome c (8.73-fold). Enhanced generation of intracellular ROS correlated well with the decreased levels (~60 %) of intracellular GSH. Sensitizing neuroblastoma cells for induction of apoptosis by p-CA identified p53-mediated upregulated accumulation of caspase-8 messenger RNA (2.8-fold). Our data report on autophagy, representing an additional mechanism of p-CA to induce growth arrest, detected by immunoblotting and fluorescence, correlated with accumulation of elevated levels (1.2-fold) of the LC3-II protein and acridine orange-stained autophagosomes, both autophagy markers. The present study indicates p-CA was effective in production of ROS-dependent mitochondrial damage-induced cytotoxicity in N2a cells.

Development and validation of an immunochromatographic assay for rapid detection of fumonisin B1 from cereal samples.

Fumonisins are one of the most agriculturally significant environmental toxins produced by Fusarium and Aspergillus species that grow on agricultural commodities in the field or during storage. Cereals contaminated with fumonisins causes serious loss to agricultural produce leads to health problems in humans and other farm animals. In the present study, polyclonal hyperimmune sera was raised against FB1 in rabbits immunized with FB1-keyhole limpet haemocyanin (KLH). Purified antibodies were used to establish a sensitive gold nanoparticle based immunochromatographic strip (ICG) for detecting FB1 levels in cereal grains. Effective on-site detection of FB1 was achieved by developing a rapid and sensitive pAb based ICG strip. This strip had a detection limit of 5 ng mL(-1) for FB1 in cereal samples and it could be completed within 3 min. Close examination of 150 cereal samples by ICG strip method revealed that 77 were fumonisin-positive. Results obtained by the developed method was further validated with well standardized HPLC method and results of strip method was correlated well with those obtained by HPLC method. In conclusion, the developed method was a better alternative for onsite detection of FB1 in cereal samples intended for human consumption to reduce risk of humans and other farm animals. The high level of FB1 concentrations recorded in present study warrants the need to develop an awareness creation programme to the farmers of India for safe handling of cereal grains at the time of harvesting and storage of grains.

Rosmarinic acid mediated neuroprotective effects against H2O2-induced neuronal cell damage in N2A cells.

AIMS: Oxidative stress plays a key role in several ailments including neurodegenerative conditions. The aim of the study was to demonstrate the effect of rosmarinic acid (RA) in preventing oxidative stress related death of neuronal cell lines. MAIN METHODS: In the present study, we demonstrated direct neuroprotective effect of RA using H2O2-induced oxidative challenge in N2A mouse neuroblastoma cells. The mechanism of neutralization of H2O2-induced toxicity by RA was evaluated using MTT, lactate dehydrogenase, mitochondrial membrane potential (MMP), intracellular ROS, and comet assays. Up-regulation of brain neuronal markers at molecular level was performed by RT-PCR. KEY FINDINGS: Results presented in the paper indicate that H2O2-induced cytotoxicity in N2A cells was suppressed by treatment with RA. Moreover, RA is very effective in attenuating the disruption of lactate dehydrogenase, mitochondrial membrane potential and intracellular ROS. Pretreatment with RA significantly prevents genotoxicity (3.7-fold, p<0.01) and promotes the up-regulation of tyrosine hydroxylase (TH) (4.5-fold, p<0.01), and brain-derived neurotrophic factor (BDNF) genes (5.4-fold, p<0.01) against H2O2-induced cytotoxicity in N2A cells. SIGNIFICANCE: Our results revealed that N2A cells are suitable cellular models to evaluate neuroprotective effects of RA, and suggest that RA may potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress.

Neuroprotective effects of bikaverin on H2O2-induced oxidative stress mediated neuronal damage in SH-SY5Y cell line.

The generation of free radicals and oxidative stress has been linked to several neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease, and Amyotrophic lateral sclerosis. The use of free radical scavenging molecules for the reduction of intracellular reactive oxygen species is one of the strategies used in the clinical management of neurodegeneration. Fungal secondary metabolism is a rich source of novel molecules with potential bioactivity. In the current study, bikaverin was extracted from Fusarium oxysporum f. sp. lycopersici and its structural characterization was carried out. Further, we explored the protective effects of bikaverin on oxidative stress and its anti-apoptotic mechanism to attenuate H2O2-induced neurotoxicity using human neuroblastoma SH-SY5Y cells. Our results elucidate that pretreatment of neurons with bikaverin attenuates the mitochondrial and plasma membrane damage induced by 100 µM H2O2 to 82 and 26% as evidenced by MTT and LDH assays. H2O2 induced depletion of antioxidant enzyme status was also replenished by bikaverin which was confirmed by Realtime Quantitative PCR analysis of SOD and CAT genes. Bikaverin pretreatment efficiently potentiated the H2O2-induced neuronal markers, such as BDNF, TH, and AADC expression, which orchestrate the neuronal damage of the cell. The H2O2-induced damage to cells, nuclear, and mitochondrial integrity was also restored by bikaverin. Bikaverin could be developed as a preventive agent against neurodegeneration and as an alternative to some of the toxic synthetic antioxidants.

Zearalenone induced toxicity in SHSY-5Y cells: The role of oxidative stress evidenced by N-acetyl cysteine.

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24h time points. DAPI staining showed apoptotic nuclei at 12 and 24h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.

Anti-apoptotic mechanism of Bacoside rich extract against reactive nitrogen species induced activation of iNOS/Bax/caspase 3 mediated apoptosis in L132 cell line.

Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India.

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: an in vivo and in vitro evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Preparations of Orthosiphon diffusus (Benth.) have been used by folk medicinal practitioners in the Western Ghats of India for treating inflammation, hepatitis and jaundice for many years and their effectiveness is widely acclaimed among the tribal communities. AIM OF THE STUDY: To evaluate the mechanisms behind the antioxidant and hepatoprotective potential of Orthosiphon diffusus methanol active fraction (MAF) using in vivo (rat) and in vitro (cell culture) models. MATERIALS AND METHODS: Neutralization of CCl4-induced hepatotoxicity by MAF was evaluated in rats. Towards this, serum levels of hepatic injury markers (lactate dehydrogenase and alkaline phosphatase), antioxidant enzymes in the liver homogenates, and histological examination were performed. In in vitro studies, mechanisms of neutralization of H2O2-induced toxicity by MAF using MTT, Comet assay and up-regulation of antioxidant enzymes at genetic level (RT-PCR) was performed in HepG2 cells. RESULTS: Rats pre-treated with Orthosiphon diffusus MAF demonstrated significantly reduced levels of serum LDH (1.3-fold, p<0.05) and ALP (1.6-fold, p<0.05). Similarly, multiple dose MAF administration demonstrated significantly enhanced levels (p<0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl4-induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H2O2-induced oxidative stress, genotoxicity and significantly enhanced (~6-fold, p<0.01) expression of genes for antioxidant enzymes. CONCLUSIONS: Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl4-induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H2O2-induced oxidative stress was completely neutralized by MAF through enhanced expression of genes for antioxidant enzymes. Therefore, this study validates the use of Orthosiphon diffusus by folk medicinal practitioners in India. Further, MAF of Orthosiphon diffusus can serve as a strong candidate for the development of herbal hepatoprotective agents.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.