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Simple Summary: We developed a new test to detect Babesia aktasi, a parasite that infects goats, using a method called semi-nested PCR. This method focuses on a specific part of the parasite's DNA to ensure accuracy. We checked the test against several other similar parasites to make sure it only detected B. aktasi, which it did successfully. To see how sensitive our test is, we used blood samples with known amounts of the parasite and found that our test could detect even very low levels of infection. Our results show that this new test is both highly accurate and sensitive, making it a valuable tool for identifying B. aktasi infections in goats. This new PCR method provides a reliable tool for detecting B. aktasi in goats, which is crucial for managing and preventing the spread of this infection, ultimately protecting goat health and improving agricultural productivity. We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10−8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

This study developed a real-time polymerase chain reaction (real-time PCR) method for the detection of Lactobacillus kefiranofaciens subsp. kefiranofaciens. Based on the 16S rDNA gene sequence and whole genome sequence of the type strain of L. kefiranofaciens subsp. kefiranofaciens ZW3, specific primers were designed and screened. The fluorescent dye SYBR Green I was used in the real-time PCR method. Its specificity, sensitivity and repeatability were evaluated, and this method was applied to detect several strains of this species and its mixtures with other lactic acid bacteria. The results showed that the proposed method was highly specific, sensitive and repeatable. The standard curve was linear with a determination coefficient (R2) of 0.965. Moreover, this method could specifically detect L. kefiranofaciens subsp. kefiranofaciens from its mixture with other lactic acid bacteria. In summary, the real-time PCR method could quickly and accurately detect L. kefiranofaciens subsp. kefiranofaciens, providing a new method for the specific qualitative and quantitative detection of L. kefiranofaciens subsp. kefiranofaciens.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10−8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity.

Different genotypes of infectious bronchitis virus (IBV) in poultry are circulating worldwide. The most efficient classification system for IBV genotypes is based on the complete sequencing of S1 gene, and the target organ can be trachea, kidney, oviduct, lung, esophagus, proventriculus, intestine, liver, spleen, bursa, cecal tonsils, cloaca, and testis. In Iran, IBV genotyping is usually performed by partial sequencing of S1 gene, and the trachea is often selected for sampling. This study applies a different genotyping method, and compares the results with the routine method of genotyping. Samples were collected from the trachea and kidneys of 50 broiler flocks with respiratory symptoms. The presence of IBV was confirmed by a real-time RT-PCR analysis targeting the 5’UTR region of the genome. Genotype of positive samples was determined by two methods of partial S1 gene sequencing and genotype-specific primers. In the real-time RT-PCR test, 88% and 90% of tracheal and kidney samples showed positive results, respectively. When the S gene was sequenced, Variant 2 (GI-23) (68.18%), 793/B (GI-13) (22.73%), Massachusetts (GI-1) (6.82%), and QX (GI-19) (2.27%) were detected in the tracheal samples, whereas QX (GI-19) and Massachusetts (GI-1) were not diagnosed in the kidney samples. In the genotypespecific method, IBV genotypes Variant 2, 793/B, Massachusetts, and QX were detected in both tracheal and kidney samples. The results of genotype-specific method also demonstrated that 70% of tracheal samples and 62% of kidney samples were infected with a single IBV genotype, while 30% of tracheal samples and 38% of kidney samples were coinfected with different IBV types. When comparing two genotyping methods, the use of genotype-specific primers was superior to partial S1 gene sequencing, not only in detecting different IBV types, but also in efficiently applying them in both trachea and kidney. [ABSTRACT FROM AUTHOR]

Background and purpose: There are various methods for molecular detection of SARS-CoV2 genome among which, PCR-based methods are the most reliable for making diagnosis. The majority of approved PCR kits for detection of Coronavirus are based on TaqMan real-time PCR which is expensive due to incorporating fluorescent and quencher-harboring probe. The aim of this study was to design a simple and affordable method for detection of SARS-CoV2 based on a more affordable SYBR green qPCR method. Materials and methods: Specific primers were designed for the virus nucleocapsid gene and the human control gene using Oligo software. The specificities of the primers were examined by Primer-BLAST capability according to NCBI database. Nasal swab samples were obtained from 10 PCR-confirmed COVID-19 patients and 10 healthy control people. Results: The designed reactions were performed in full compliance with the kit approved by the Ministry of Health to discriminate healthy individuals from COVID-19 patients. The Ct cut-off values calculated for N1 and N2 reactions were 39.72 and 39.69, respectively. Conclusion: The designed SYBR green-based reaction showed a potential role for detection of SARS-CoV2 genome in clinical samples and this method can be considered as a less-expensive alternative to TaqMan real time PCR if the primers and standard reaction conditions are properly designed.

Nerium oleander is one of the most poisonous plants, and its accidental ingestion has frequently occurred in humans and livestock. It is vital to develop a rapid and accurate identification method for the timely rescue of oleander-poisoned patients and the investigation of poisoning cases. In this study, a specific and highly sensitive quantitative real-time PCR (qPCR)-based method was developed to identify oleander in mixture systems and simulated forensic specimens (SFS). First, a new pair of oleander-specific primers, JZT-BF/BR, was designed and validated. Then, a qPCR method was developed using the primers, and its detective sensitivity was examined. The results showed that JZT-BF/BR could specifically identify oleander in forage and food mixtures, and qPCR was capable of accurate authentication even at a low DNA concentration of 0.001 ng/μL. This method was further applied to the analysis of SFS containing different ratios of N. oleander. The method was confirmed to be applicable to digested samples, and the detection limit reached 0.1% (w/w) oleander in mixture systems. Thus, this study undoubtedly provides strong support for the detection of highly toxic oleander and the diagnosis of food poisoning in humans and animals. [ABSTRACT FROM AUTHOR]

Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/μL of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccusneobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays. [ABSTRACT FROM AUTHOR]

Abstract Background The dry body of the Tokay Gecko (Gekko gecko) is the source of a valuable traditional Chinese medicine, it is therefore listed as a Class II protected animal species in China. Due to increasing market demand and a declining supply of the species, a considerable number of adulterants have emerged in the market. Thus, it is necessary to establish an accurate and rapid method of identification for distinguishing G. gecko from its adulterants and for separating it from highly processed products. Methods A total of 274 COI sequences were analyzed by using MEGA 5.0 software. Several specific primers were designed to amplify mini-barcode regions and identify G. gecko from its counterfeits and products. Results 274 COI sequences of G. gecko and 15 adulterants species were analyzed. G. gecko could be distinguished from its adulterants through BLAST analysis, intra- and inter-specific distance analyses, and an NJ tree based on COI sequences. Two pairs of specific primers designed for this study, COISF2/COISR2 and COISF3/COISR3, amplified 200- and 133-bp fragments of the COI region, respectively, both of which were suitable for the identification of G. gecko and its adulterants. Furthermore, COISF3/COISR3 detected G. gecko in 15 batches of products. Conclusion Therefore, the specific DNA mini-barcoding method developed here may be a powerful tool for the identification of G. gecko and counterfeits, and may also be used to distinguish G. gecko from its highly processed by-products.

The article focuses on real-time PCR detection and quantification of Fusarium spp., potential producer of class B fumonisin mycotoxins in mature corn kernels using primers designed to genes FUM1 and FUM6, involved in mycotoxin biosynthesis. Research have shown that the real-time PCR assay can be used for detecting and quantifying potentially mycotoxigenic Fusarium in the maize kernels by the genes involved in mycotoxin synthesis. Three pairs of primers specific to genes involved in the synthesis of mycotoxin FB known to contaminate maize and maize products were developed and a pair of primers specific to a conserved Fusarium beta-tubulin gene, aimed at detection of most Fusarium species. There has been applied quantitative PCR assay for quantification of total Fusarium spp. and potentially mycotoxigenic Fusarium verticillioides.

Background and purpose: There are various methods for molecular detection of SARS-CoV2 genome among which, PCR-based methods are the most reliable for making diagnosis. The majority of approved PCR kits for detection of Coronavirus are based on TaqMan real-time PCR which is expensive due to incorporating fluorescent and quencher-harboring probe. The aim of this study was to design a simple and affordable method for detection of SARS-CoV2 based on a more affordable SYBR green qPCR method. Materials and methods: Specific primers were designed for the virus nucleocapsid gene and the human control gene using Oligo software. The specificities of the primers were examined by Primer- BLAST capability according to NCBI database. Nasal swab samples were obtained from 10 PCRconfirmed COVID-19 patients and 10 healthy control people. Results: The designed reactions were performed in full compliance with the kit approved by the Ministry of Health to discriminate healthy individuals from COVID-19 patients. The Ct cut-off values calculated for N1 and N2 reactions were 39.72 and 39.69, respectively. Conclusion: The designed SYBR green-based reaction showed a potential role for detection of SARS-CoV2 genome in clinical samples and this method can be considered as a less-expensive alternative to TaqMan real time PCR if the primers and standard reaction conditions are properly designed. [ABSTRACT FROM AUTHOR]

Soybean is threatened by many pathogens that negatively affect this crop's yield and quality, such as various Fusarium species that cause wilting and root rot diseases. Fusarium root rot (FRR) in soybean can be caused by F. graminearum and other Fusarium spp. that are associated with Fusarium head blight (FHB) in cereals. Therefore, it was important to inquire whether Fusarium pathogens from soybean can cause disease in wheat and vice versa. Here, we investigated the FRR complex in Manitoba (Canada) from symptomatic plants, using both culture- and molecular-based methods. We developed a molecular diagnostic toolkit to detect and differentiate between several Fusarium spp. involved in FHB and FRR, then we evaluated cross-pathogenicity of selected Fusarium isolates collected from soybean and wheat, and the results indicate that isolates recovered from one host can infect the other host. Trichothecene production by selected Fusarium spp. was also analyzed chemically via liquid chromatography mass spectrometry in both soybean (root) and wheat (spike) tissues. Trichothecenes were also analyzed in soybean seeds from plants with FRR to check the potentiality of trichothecene translocation from infected roots to the seeds. All of the tested Fusarium isolates were capable of producing trichothecenes in wheat spikes and soybean roots, but no trichothecenes were detected in soybean seeds. This study provided evidence, for the first time, that trichothecenes were produced by several Fusarium spp. (F. cerealis, F. culmorum, and F. sporotrichioides) during FRR development in soybean. [ABSTRACT FROM AUTHOR]

Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/μL of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccusneobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays.

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract The gypsy moth—Lymantria dispar (Linnaeus)—is a worldwide forest defoliator and is of two types: the European gypsy moth and the Asian gypsy moth. Because of multiple invasions of the Asian gypsy moth, the North American Plant Protection Organization officially approved Regional Standards for Phytosanitary Measures No. 33. Accordingly, special quarantine measures have been implemented for 30 special focused ports in the epidemic areas of the Asian gypsy moth, including China, which has imposed great inconvenience on export trade. The Asian gypsy moth and its related species (i.e., Lymantria monocha and Lymantria xylina) intercepted at ports are usually at different life stages, making their identification difficult. Furthermore, Port quarantine requires speedy clearance. As such, it is difficult to identify the Asian gypsy moth and its related species only by their morphological characteristics in a speedy measure. Therefore, this study aimed to use molecular biology technology to rapidly identify the Asian gypsy moth and its related species based on the consistency of mitochondrial DNA in different life stages. We designed 10 pairs of specific primers from different fragments of the Asian gypsy moth and its related species, and their detection sensitivity met the need for rapid identification. In addition, we determined the optimal polymerase chain reaction amplification temperature of the 10 pairs of specific primers, including three pairs of specific primers for the Asian gypsy moth (L. dispar asiatic), four pairs of specific primers for the nun moth (L. monocha), and three pairs of specific primers for the casuarina moth (L. xylina). In conclusion, using our designed primers, direct rapid identification of the Asian gypsy moth and its related species is possible, and this advancement can help improve export trade in China.

Introduction: Canola is one of the important oil crops in Iran and blackleg disease caused by Leptosphaeriamaculans is an economically important disease of rapeseed especially in the northern provinces of Iran. At the beginning of the season in early autumn, L. maculansareinitiatesby air-borne ascospores released from infected stubbles of previous crops. These ascospores germinate and produce leaf lesions. The fungus then grows systemically from the leaf lesions to stem where cankers are producedwhich can result in major yield loss. Blacklegiscaused by a complex of at least two species of Leptosphaeria: L. maculans and L. biglobosa. Of these two species whichL. maculanswasis much more prevalent and virulent than L. biglobosa and was reported from almost all oilseed rape growing regions of the world.A number ofmethods has been developed to differentiate isolates of thesetwo groupsthrough morphological, physiological, genetic and molecular markers. Among these virulence markers of group pathogenicity are most widely used for characterizing the pathogen population. The purpose of this research work was to investigate isolates identification based on colony morphology and molecular methods. Moreover, for management strategies to be effectiveit is important to recognize the population structure based on pathogenicity groups and an understanding of possible shift in fungus population in thenorth of Iran. Materials and Methods: Infected plants of oilseed rape were collected from the northeast Iranduring 2013-2016. Diseased plant organs with clearly defined symptoms of the disease were used for the isolation of the fungi. All isolates were analyzed using a combination of morphological and physiological. In order to confirm the identification of L. maculans from oilseed rape isolates, DNA was prepared using the standard method described previously. The ITS region of genome of all isolates was amplified using polymerase chain reaction with specific primers pair LmF, lmR. Some isolates were sequenced with ITS1 and ITS2 along with the 5.8S rRNAregion and then sequence data were compared with that of available L. maculans isolates in Gene Bank. Aggressive isolates of L. maculans collected in widely separated geographic regions were further divided into pathogenicity groups based on virulence of three differentBrassica napuscultivars. Results and Discussion: Morphological and physiological and molecular characterizations of 72 isolates were performed. Isolates grewslowly, the pycnidia of the fungus were black, globose to subglobose in shape and conidia were single-celled, hyaline. The most of isolates formed yellow and pigment formation was scored in six groups in PDB at 18°C in the dark on the shaker. The PCR detection showed that all isolates were amplified by L. maculans-specific primer pair and a 334-bp PCR product was reliably amplified from L. maculans. The sequence analysis of the ITS region revealed that the sequences had 99.7% sequence similarity with the ITS sequences of known L. maculansisolates in GenBank by NCBI Blast search. This isolates were registered with accession number KX649997 and KX792142 in gene bank and this confirmed that the pathogen exists in Brassica napus samples. The role of morphological identification in virulence is complex and the production of pigment and mycelial growth is not always correlated with virulence. Therefore, based on amplification with type specific primers the amplified product fragment specific for group A was found in all isolates. It is important to know which pathotype is present, because the pathotypes of L. maculans differ in the amount of damage they cause. Based on the reaction of isolates on differential cultivars all four group pathogenicity PG-2, PG-3, PG-4 and PGT were observed in aggressive isolates. The most isolates were classified to be a pathogenicity PG-4 virulent on three differentcultivars and for the first time wasidentified in Iran. Conclusions: In order to determine whether isolates belonging to the canker L. maculans are present in the north of Iran, initial species identification based on colony morphology was confirmed using molecular methods. Asthe pathogen causes considerable losses, thefast identification and pathotype determination are important for agriculture and successful management of blackleg disease. Our results showed that an understanding of possible shift in fungus populations of PG2 to PG4 will be of value in developing strategies for successful management of blackleg disease.

Eucalyptus wilt disease was one of the most important stem disease in Eucalyptus plantation. Fusarium solani was one of the main pathogen. A specific fragment was detected from F. solani based on the RAPD-PCR technique. It was then cloned and sequenced. Specific SCAR primers F9/R9 were designed according to the sequencing results. The amplification conditions were optimized and the primer sensitivity was detected. With the SCAR primer F9/R9, a clear and bright fragment of about 200 bp was amplified only from F. solani, but no fragment from other pathogen. The primer could detect the specific fragment from 4×10-7 ng·μL-1 of genomic DNA and individual spore. F. solani from Eucalyptus infection stem and soil could be detected by these SCAR primer. Thus, the SCAR primer F9/R9 could be used for early diagnosis and quarantine of Eucalyptus wilt disease. This method was simple and of high specificity. [ABSTRACT FROM AUTHOR]

A quantitative PCR (qPCR)-assay was developed to detect airborne inoculum of Nothopassalora personata, causal agent of late leaf spot (LLS) on peanut, collected with a modified impaction spore trap. The qPCR assay was able to consistently detect as few as 10 spores with purified DNA and 25 spores based on crude DNA extraction from rods. In 2019, two spore traps were placed in two peanut fields with a history of LLS. Sampling units were replaced every 2 to 4 days and tested with the developed qPCR assay, while plots were monitored for symptom development. The system detected inoculum 35 to 56 days before visual symptoms developed in the field, with detection related to environmental parameters affecting pathogen life-cycle and disease development. This study develops the framework of the qPCR spore trap system and represents the initial steps towards validation of the performance of the system for use as a decision support tool to complement integrated management of LLS.

Red leaf blotch of almond, caused by the fungus Polystigma amygdalinum, results in early defoliation of trees and decreases fruit production in many Mediterranean and Middle Eastern countries. A latent period of 30-40 d has been reported for this pathogen before disease symptoms are expressed, which makes targeted fungicide control difficult. A quantitative real-time PCR detection method was developed to detect and quantify the fungus in biological samples, to assist early detection. A primer pair was designed based on the ITS region of the fungal rDNA, and this was highly specific and sensitive, enabling detection of a minimum of 12 pg of P. amygdalinum DNA and seven ascospores in artificially-prepared ascospore suspensions. A protocol was also developed to quantify ascospores on plastic tapes which are commonly used in volumetric air samplers. The detection limit for these samples was the same as for ascospore quantification in aqueous suspensions. The pathogen was also quantified in naturally infected leaves showing different stages of disease development, including early stages of leaf infection with doubtful visual identifications. Future practical applications of the method developed here are discussed in view of improving the management of red leaf blotch of almond. [ABSTRACT FROM AUTHOR]

Bacteriocins are ribosomally synthesized proteins/peptides that have bacteriocidal or bacteriostatic trait against genitically closed species. Bacteriocins have role as biopreservative and increase of the safety of food products. Study on native strains of lactobacillus plantarum (DL2 ،BL1 ،L28 ،L27 ، L25 and EL3) that their growth inhibition ability had been analyzed, was performed. after approving inhibition ability of strains by disk diffusion in previous study, to investigate bacteriocin encoding gene, polymerase chain reaction (PCR) was performed by specific primers. The correspondant region was amplified in L25, BL1, EL3 and L28 strains. Using designed primers and sequencing could be shown that EL3 strain contained the structural genes for the bacteriocins plnEF, plnJK and plnN, howover, the Lb. plantarum strain BL1 and L28 contained only the structural genes for plantaricin EF production. sequences showed ٪99 similarity with bacteriocin genes of Lactobacillus plantarum strains recorded in Gene Bank database. The sequences were submitted at NCBI with accession numbers of EL3 (KT028600), L28 (KT028601) and BL1 (KT028602). [ABSTRACT FROM AUTHOR]

Among oysters, species of Crassostrea (Sacco, 1897) are the most attractive to aquaculture. In Brazil, the genus is represented by C. rhizophorae (Guilding, 1828) and C. brasiliana (Lamarck, 1819). Because the maturation and breeding technology is not well developed for these species, aquaculturists need a reliable method to decide the correct time to place spat collectors in the field, and to identify both species, which are morphologically similar. In this study a specific Multiplex PCR protocol was developed, using one pair of universal primers from 18S rDNA as a positive control and a pair of specific primers for each target species. The sensitivity and specificity of the protocol was evaluated. It detected C. rhizophorae DNA in low concentrations, and C. brasiliana DNA in even lower concentrations. Further, the Multiplex PCR proved efficient in detecting DNA in concentrations equivalent to that of a single larva of each species, either separated or combined, when mixed with total DNA extract of a plankton sample representing 1000 L of filtered water. Field tests confirmed the applicability of the protocol, which holds the promise to become an important tool for aquaculture or conservation programs, allowing for the continuous monitoring of the life cycle of C. brasiliana and C. rhizophorae, by detecting the right periods of larval release and settlement.

Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease in alfalfa. The isolates of C. trifolii were obtained from diseased alfalfa stems collected from field in Serbia. It was determined by pathogenicity examination that four isolates (Luc-7, Luc-17, Luc-27, Luc-33) can cause stem lesions on inoculated alfalfa plant. Our isolates were compared using reference isolates of C. trifolii (CBS 158.83). Isolates on MA and CDA developed olive green to grey colonies with white margin, while the substrate got dark olive green color. Conidiophores were hyaline, varied in length and produced a succession of conidia apically. Conidia were hyaline, straight, rounded at the ends, and non-septated with average size 7.85x3.87 μm. Average sizes of appressoria were 7.5-16.5 x 5.5 x 8.9 μm. The polymerase chain reaction (PCR) with one set of specific primers was used for the detection of examined isolates of Colletotrichum trifolii. Amplification of desired DNA fragment (590 bp) was determined using specific primer pair TB3-F/TB3-R. Achieved results of amplification indicated that the isolates Luc-7, Luc-17, Luc-27, as well as CBS 158.83, had traits of C. trifolli. Amplification and band on about 430 bp appeared in isolate Luc-33, which also belonged to C. trifolli species.

Background: Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity. Results: Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species. Conclusions: The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/. [ABSTRACT FROM AUTHOR]

A survey of bleeding canker disease, caused by Pseudomonas syringae pv. aesculi, was undertaken across Ireland. Incidence has become severe and can be considered epidemic, as 61% of the 1587 horse chestnut trees surveyed showed symptoms of the disease. Bacteria were isolated from a sample of trees and characterized using gyrB DNA sequencing. DNA was also extracted directly from wound tissue. The Irish P. syringae pv. aesculi genotype was identical to genotypes previously sequenced with gyrB from the UK and some other locations in Europe. Real-time PCR, using existing primers and a newly designed, more pathovar-specific primer set, was assessed for use in disease screening. With molecular screening, a total of 11 trees from a sample of 55 tested positive for P. syringae pv. aesculi in Ireland. It was more efficient to extract DNA directly from wound tissue, especially fresh bark, for disease detection than to undertake bacterial isolation with subsequent molecular analysis. A further set of sequencing primers was developed for the amplification of the gyrB gene from P. syringae pv. aesculi and their specificity was shown using a diverse sample of bacterial isolate DNAs. The study also isolated and identified other bacterial species from diseased material; some of these are known pathogens ( Brenneria nigrifluens, P. marginalis and P. syringae) or have previously been identified as potentially beneficial endophytes of host trees ( Erwinia billingiae, E. tolentana, P. fluorescens, P. putida and Raoultella). [ABSTRACT FROM AUTHOR]

Amplified Fragment Length Polymorphism molecular markers (AFLPs) has been developed combining procedures of RFLPs and RAPDs molekular markers, i.e. the first step is restriction digestion of the genomic DNA that is followed by selective amplification of the restricted fragments. The advantage of the AFLP technique is that it allows rapid generation of a large number of reproducible markers. The reproducibility of AFLPs markers is assured by the use of restriction site-specific adapters and adapter-specific primers for PCR reaction. Only fragments containing the restriction site sequence plus the additional nucleotides will be amplified and the more selected nucleotides added on the primer sequence the fewer the number of fragments amplified by PCR. The amplified products are normally separated on a sequencing gel and visualized after exposure to X-ray film or by using fluorescent labeled primers. AFLP shave proven to be extremely proficient in revealing diversity at below the species level. A disadvantage of AFLP technique is that AFLPs are essentially a dominant marker system and not able to identify heterozygotes.