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Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technique that has been adopted for simple, robust, rapid, reliable diagnostics of nematodes. In this study, the real-time RPA assay and RPA assay combined with lateral flow dipsticks (LF-RPA) have been developed targeting the ITS rRNA gene of the British root-knot nematode, Meloidogyne artiellia. The assay provided specific and rapid detection of this root-knot nematode species from crude nematode extracts without a DNA extraction step with a sensitivity of 0.125 second-stage juvenile (J2) specimen per a reaction tube for real-time RPA during 11 min and a sensitivity of 0.5 J2 specimens per a reaction tube for LF-RPA during 25 min. The RPA assays were validated with a wide range of non-target root-knot nematodes. The LF-RPA assay has great potential for nematode diagnostics in the laboratory having minimal available equipment.

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Rapid and reliable diagnostic methods for plant-parasitic nematodes are critical for facilitating the selection of effective control measures. A diagnostic recombinase polymerase amplification (RPA) assay for Aphelenchoides fragariae using a TwistAmp® Basic Kit (TwistDx, Cambridge, UK) and AmplifyRP® Acceler8® Discovery Kit (Agdia, Elkhart, IN, USA) combined with lateral flow dipsticks (LF) has been developed. In this study, a LF-RPA assay was designed that targets the ITS rRNA gene of A. fragariae. This assay enables the specific detection of A. fragariae from crude nematode extracts without a DNA extraction step, and from DNA extracts of plant tissues infected with this nematode species. The LF-RPA assay showed reliable detection within 18-25 min with a sensitivity of 0.03 nematode per reaction tube for crude nematode extracts or 0.3 nematode per reaction tube using plant DNA extracts from 0.1 g of fresh leaves. The LF-RPA assay was developed and validated with a wide range of nematode and plant samples. Aphelenchoides fragariae was identified from seed samples in California. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment.

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Meat adulteration have become a global issue, which has increasingly raised concerns due to not only economic losses and religious issues, but also public safety and its negative effects on human health. Using optimal primers for seven target species, a multiplex PCR method was developed for the molecular authentication of camel, cattle, dog, pig, chicken, sheep and duck in one tube reaction. Species-specific amplification from the premixed total DNA of seven species was corroborated by DNA sequencing. The limit of detection (LOD) is as low as 0.025 ng DNA for the simultaneous identification of seven species in both raw and heat-processed meat or target meat: as little as 0.1% (w/w) of the total meat weight. This method is strongly reproducible even while exposed to intensively heat-processed meat and meat mixtures, which renders it able to trace meat origins in real-world foodstuffs based on the authenticity assessment of commercial meat samples. Therefore, this method is a powerful tool for the inspection of meat adulterants and has broad application prospects.

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Frequent meat frauds have aroused significant social attention. The aim of this study is to construct a two-tube hexaplex polymerase chain reaction (PCR) method offering accurate molecular authentication of twelve meat species in actual adulteration event. Deoxyribonucleic acid (DNA) sequencing demonstrates that designed primers can specifically amplify target species from genomic DNA mixture of six species in each tube reaction, which showed 100% accuracy of horse (148 bp), pigeon (218 bp), camel (283 bp), rabbit (370 bp), ostrich (536 bp), and beef (610 bp) as well as turkey (124 bp), dog (149 bp), chicken (196 bp), duck (277 bp), cat (380 bp), and goose (468 bp). A species-specific primer pair produced the target band in the presence of target genomic DNA but not non-target species. Through multiplex PCR assays with serial concentration of the DNA mixture of six species in each PCR reaction, the detection limit (LOD) of the two-tube hexaplex PCR assay reached up to 0.05-0.1 ng. Using genomic DNA isolated from both boiled and microwave-cooked meat as templates, PCR amplification generated expected PCR products. These findings demonstrate that the proposed method is specific, sensitive and reproducible, and is adequate for food inspection. Most importantly, this method was successfully applied to detect meat frauds in commercial meat products. Therefore, this method is of great importance with a good application foreground.

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The genus Laccaria (Hydnangiaceae, Agaricales) plays an important role in forest ecosystems as an ectomycorrhizal fungus, contributing to nutrient cycles through symbiosis with many types of trees. Though understanding Laccaria diversity and distribution patterns, as well as its association with host plants, is fundamental to constructing a balanced plant diversity and conducting effective forest management, previous studies have not been effective in accurately investigating, as they relied heavily on specimen collection alone. To investigate the true diversity and distribution pattern of Laccaria species and determine their host types, we used four different approaches: specimen-based analysis, open database search (ODS), NGS analysis, and species-specific PCR (SSP). As a result, 14 Laccaria species have been confirmed in Korea. Results regarding the species distribution pattern were different between specimen-based analysis and SSP. However, when both were integrated, the exact distribution pattern of each Laccaria species was determined. In addition, the SSP revealed that many Laccaria species have a wide range of host types. This study shows that using these four different approaches is useful in determining the diversity, distribution, and host of ECM fungi. Furthermore, results obtained for Laccaria will serve as a baseline to help understand the role of ECM fungi in forest management in response to climate change.

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Meloidogyne luci has been identified in various countries around the world parasitizing economically important crops and, due to its potential to cause serious damage to agriculture, was included in the European and Mediterranean Plant Protection Organization Alert List in 2017. This species shares morphological and molecular similarities with M. ethiopica and M. inornata, and a M. ethiopica group was therefore established. Although specific primers for the DNA amplification of species belonging to the M. ethiopica group have been developed previously, the primers were not species-specific, so molecular markers for the specific detection of M. luci are still needed. The objective of this study was to develop a SCAR marker for the detection of M. luci and the discrimination from other Meloidogyne spp. based on the intraspecific variability found in RAPD markers. RAPD screening of M. luci and M. ethiopica genome was used for the identification of a specific amplification product on M. luci, which was cloned, sequenced and converted into a SCAR marker. The specificity of the designed primers (Mlf/r) was tested and produced a fragment (771 bp) for all nine M. luci isolates with no amplification for the other nine Meloidogyne spp., including M. ethiopica and M. inornata. Additionally, the proper amplification of the M. luci SCAR-marker was also successful with DNA from galls of M. luci infected tomato roots. The results obtained in this study reveal that the specific molecular detection of M. luci was achieved and that the developed methodology can be used for routine diagnosis purposes, which are essential to monitoring the distribution and spread of M. luci in order to implement future effective and integrated nematode pest management programs.

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BACKGROUND: Bacteriocins are proteinaceous compounds produced from lactic acid bacteria. Bacteriocins are well-known for their antibacterial potential and safety for application in food. However, the commercial availability of bacteriocin is facing several limitations; among them is the low yield and short stability period. That calls for a new strategy for overcoming these hurdles. Among these approaches is incorporating bacteriocin in nanoparticles. So, the aim of this study was to enhance the plantaricin produced from isolated Lactobacillus plantarum strain using nanotechnology. RESULTS: In this study, the plnEF genes encoding plantaricin EF have been identified and sequenced (accession number of MN172264.1). The extracted bacteriocin (EX-PL) was obtained by the ammonium sulfate method. Then, it was used for biosynthesizing plantaricin-incorporated silver nanoparticles (PL-SNPs). The synthesized nanoparticles were confirmed by SEM-EDAX analysis. The antibacterial activity of both combined (PL-SNPs) and extracted plantaricin (EX-PL) were tested against some strains of foodborne pathogenic bacteria. The results revealed that the antibacterial activities were increased by 99.2% on the combination of bacteriocin with the silver nanoparticle. The MIC of EX-PL (7.6 mg/mL) has been lowered after incorporating into silver nanoparticles and reached 0.004 mg/mL for PL-SNPs. Despite that extracted plantaricin showed no inhibitory activity towards Listeria monocytogenes, plantaricin-incorporated silver nanoparticles displayed inhibitory activity against this strain. Furthermore, the stability period at 4 °C was increased from 5 days to 60 days for EX-PL and PL-SNPs, respectively. CONCLUSIONS: Plantaricin-incorporated silver nanoparticles possess higher antibacterial activity and more stability than the free one, which makes it more fitting for combating foodborne pathogens and open more fields for applications in both food and pharmaceutical industries.

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The Lactobacillus casei group, which includes the closely related species L. casei, L. paracasei, L. rhamnosus, and L. chiayiensis, has been under debate regarding its taxonomy because of the difficulty in distinguishing the species from each other. In the present study, we developed a novel real-time PCR assay for distinguishing the L. casei group species. The pan-genome, as determined by the genomes of 44 strains, comprised 6789 genes, comparative genomic analysis showed that L. casei group strains were classified by species. Based on these results, species-specific genes were identified, and primers were designed from those genes. Real-time PCR clearly distinguished each species of the L. casei group and specifically amplified only to the target species. The method was applied to 29 probiotic products, and the detected results and label claims were compared. Total 23 products were in accordance with the label claims, and the remaining products contained species different from those stated in the label claims. Our method can rapidly and accurately distinguish the L. casei group species in a single reaction. Hence, our assay can be applied to identify L. casei group species from food or environmental samples and to accurately determine the nomenclature of the species.

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BACKGROUND: Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. RESULTS: To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S-23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S-23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. CONCLUSIONS: The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

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: Centrocestus formosanus is a digenetic trematode with a complex life cycle, involving invertebrate and vertebrate hosts, humans included. In particular, it causes gill lesions and mortality in freshwater fish species, and gastrointestinal symptoms in infected humans. Here, we describe the occurrence of C. formosanus infection in zebrafish imported in Italy and propose a newly designed species-specific primer pair to ameliorate the diagnostic investigations for C. formosanus. Gill arches of 30 zebrafish were examined for the presence of encysted metacercariae under a stereomicroscope and processed through molecular analyses targeting the ribosomal internal transcribed sequence 2 (ITS2). Although C. formosanus distribution was originally restricted to Asia, it has been subsequently reported in new countries, revealing itself as an invasive species and raising important concerns for biodiversity, economy, scientific research, as well as animal and public health. Given the crucial role played by the ornamental fish industry in spreading this parasite, there is an urgent need for control measures to prevent the introduction and establishment of C. formosanus in non-endemic areas, including Europe. We also suggest developing new strategies in microbiology and epidemiology to better explore this new globalization-derived invasive species.

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A rapid PCR technology was developed to differentiate Cervus antlers species and adulteration based on the difference in mitochondrial genome. Three specifically designed primer sets were confirmed to have high inter-species specificity and good intra-species stability. Limits of detection were estimated to be 1 ng of genomes for reindeer and 10 ng for the other species. Especially, when the mixture of Cervus antlers and reindeer or sambar was assayed, these primer sets still exhibited strong capability of differentiation but not the conventional COI barcoding. By using the newly developed approach, five batches out of fourteen commercial Cervus antler products were identified to be fake products made from reindeer antlers. It has shown its good potential to be extensively applied in the identification of counterfeits or adulterates of Cornu Chinese medicines for their pulverized and processed form, and even the traditional Chinese patent medicines composed of these species.

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Fasciolosis among domestic ruminants has resulted in a decrease in the production of milk products and has occasionally led to the deaths of young ruminants due to of acute infections. This study aimed to discriminate between the eggs of Fasciola gigantica and other trematode eggs in samples collected from ruminant feces specimens using PCR-based methods with the new candidate gene Cytochrome B (CYTB). A species-specific primer was developed with a high degree of sensitivity (3.285 pg). The primer was able to amplify the F. gigantica genomic DNA and there were no positive results with the other related trematodes (Paramphistomum sp., Orthocoelium sp., Fischoederius sp., Calicophoron sp., Echinostoma revolutum, E. cinetorchis, E. ilocanum and Isthmiophora hortensis), freshwater snails (Lymnaea auricularia, Bithynia siamensis, Indoplanorbis exustus, Melanoides tuberculata, Tarebia granifera) or definitive hosts (Bos primigenius and Bubalus bubalis). The minimum concentration of DNA from eggs that could be give a positive result was 3.285 pg. Moreover, the results of the study confirmed the existence of F. gigantica in Nakhon Pathom Province with a high prevalence (28.57%) and revealed the area of infection through epidemiological mapping. Thus, the species-specific primer and epidemiological data in this study may be helpful for use in epidemiological studies, phylogenetic studies and veterinary studies in the future.

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Echinostoma revolutum is known as a significant intestinal trematode in various species of animals and humans. It presents complexities in terms of both the morphological and molecular biological data. This is the first study of the application of Cytochrome B gene (CYTB) as a target for studying the phylogeny and designing species-specific primer of E. revolutum. Adult trematodes were harvested from experimentally infected hamsters at 18 days of post-infection. Each worm was identified based on their morphological appearance. The novel CYTB primers were designed from other Echinostoma species to initially amplify CYTB region in E. revolutum. All sequence data of E. revolutum in five provinces of Central Thailand were used as the target for designing the species-specific primer for E. revolutum. The results revealed that CYTB gene can separate E. revolutum into two sister groups by geographical distribution, comprising the eastern and western area groups. Moreover, it also separates E. revolutum from other Echinostoma species, including two sibling species; E. caproni and E. paraensei. In addition, we developed the high performance species-specific primer of E. revolutum. It can detect DNA from a single egg, as well as cercaria, metacercaria and adult stages of this trematode with no cross-reactions to other trematodes and their hosts. Therefore, this research is a positive initial step for the future study of E. revolutum CYTB. The future studies based on this gene should be continued with all species in revolutum complex to overcome the problems of systemic classification that arise in this complex group.

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A multiplex PCR assay was developed to simultaneously differentiate four antelope species and identify adulteration in Cornu Saigae Tataricae. Four novel primer sets were designed with high inter-species specificity and intra-species stability. Limit of detection was estimated to be 10 ng of genomes. When a mixture of antelope hornand fake species was assayed, it exhibited powerful differentiation capability. 5 out of 12 batches of commercialproducts were identified to be counterfeited or adulterated with Ovis aries Linnaeus and/or Capra hircus Linnaeus. It is readily applicable in routine analysis for identification of sham or adulterants of Cornu Saigae Tataricae.

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A novel PCR technology was developed to detect short DNA fragments using species-specific primers for rapid and non-sequencing authentication of Bombyx batryticatus based on differences in the mitochondrial genome. Three specifically designed primer reactions were established to target species for the reliable identification of their commercial products. They were confirmed to have a high inter-species specificity and intra-species stability. The limit of detection was estimated as 1 ng of genomes for Beauveria bassiana and 100 pg for Bombyx mori and Metarhizium anisopliae. Furthermore, validation results demonstrated that raw materials and their processed products can be conveniently authenticated with good sensitivity and precision using this newly proposed approach. In particular, when counterfeits were assayed, these primer sets performed well, whereas COI barcoding technology did not. These could also assist in the discrimination and identification of adulterates of other animal-derived medicines in their pulverized and processed forms and even in complexes.

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Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock protein (dnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect dnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.

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A novel non-sequencing approach was developed to detect short DNA fragments (ca 100 bp) for rapid authentication of two natural products, namely Testudinis Carapax et Plastrum and Trionycis Carapax, based on the difference in mitochondrial genome. Five specifically designed primer reactions were established to target species for reliable identification of their commercial products. They were confirmed to have a high level of inter-species-specificity and good intra-species stability. The limit of detection was estimated to be 1 ng of genomes for all of five assays. Also, the validation results demonstrated that the raw materials and processed products in addition to some of the highly processed products can be conveniently authenticated with good sensitivity and precision by this newly proposed approach. Especially, when reference sample mixtures were assayed, these primer sets have still performed well but not the prevailing COI barcoding technology. These could assist in the discrimination and identification of other animal-derived medicines for their form of raw material, the pulverized and the complex.

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In Benin, yam production continues to face numerous production constraints, including yield and quality reduction by Scutellonema bradys. Implementation of efficient management techniques against this pest requires an improved understanding, including at the molecular level, of the pest. The current study aimed at identifying the Scutellonema spp. associated with yam in Benin and investigating the phylogenetic relationships between populations. Nematodes of the genus Scutellonema were obtained from tubers exhibiting external dry rot symptoms. DNA was extracted from nematodes belonging to 138 populations collected from 49 fields from 29 villages. For 51 of these populations, both the ITS1 and COI regions could be amplified via PCR, sequenced, compared with available sequences in the NCBI database and were identified as S. bradys. Maximum likelihood was used to construct 60% consensus phylogenetic trees based on 51 sequences. This phylogenetic analysis did not reveal any genetic separation between populations by cultivar, village, cropping system nor by agroecological zone. Neither could any subgroups within S. bradys be separated, indicating that no subspecies were present. An earlier published species-specific primer set was verified with the DNA of the 51 sequences and was considered a reliable and rapid method for S. bradys identification.

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It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences. The primers were intended to be specific at the species level within relevant microbial communities, i.e., a defined genomics background. The primers were tested with samples collected from the Daqu (also called fermentation starters) and pit mud of a traditional Chinese liquor production plant. Sixteen pairs of primers were found to be suitable for identification of individual species. Among them, seven pairs were chosen to measure the abundance of microbial species through quantitative PCR. The combination of near full-length ribosome RNA gene library sequencing and Primer-Blast may represent a broadly useful protocol to quantify multiple species in complex microbial population samples with species-specific primers.

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Gelatin, a purified protein derived mostly from pig skin and bovine tissue, is used widely in both food and pharmaceutical industries. Here, to determine the species of origin of capsule gelatin, we developed a sensitive and reliable test using the polymerase chain reaction (PCR) method, which included 1) species-specific or universal primer sets, designed to detect short 16S ribosomal RNA (rRNA) gene sequences from cow, pig, and fish (tilapia) as well as genes encoding the large subunit of plant ribulose-1,5-bisphosphate carboxylase oxygenase and 2) species-specific PCR coupled with whole-genome amplification. This method was used to verify manufacturing label claims of 28 gelatin capsule samples sold as dietary supplements. The results from 27 samples were consistent with gelatin-related information on the manufacturer label, while one sample that mentioned tilapia gelatin was found to contain only bovine DNA. This rapid method can therefore be used to verify the authenticity of gelatin capsules.

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