PCR Primer Sets Research Articles

Detecting soybean and milk in dairy and soy products with post-PCR high resolution melting assays

We have developed and validated post-PCR (polymerase chain reaction) high resolution melting (HRM) based assays that allow identifying the presence of two major allergenic foods – soybean and bovine milk – in food products. A new set of primers for PCR was developed for detection of the gene encoding the soybean protein lectin. The assay was validated using reference samples and used for the analysis of artificially prepared mixed samples of dairy and soy products of different matrices, as well as of real products available in the market (spray creams). The limits of detection (LODs) of the soybean (8 copies) and bovine milk (4 copies) assays were lower compared to LODs of other previously published PCR-based assays. The analysis of several commercial spray creams revealed an undeclared presence of soybean in one of the samples. The newly developed HRM assays are precise and robust alternatives for the control of food composition and falsification by competent authorities.

Prevalence of the Helicobacter pylori babA2 Gene in Children Mainly Depends on the PCR Primer Set Used.

Various polymerase chain reaction- (PCR-) based methods with varying positivity rates were designed to detect the Helicobacter pylori babA2 gene. To compare different primer sets, babA2 prevalence was determined in 279 H. pylori-positive pediatric samples using the 832 bp, 139 bp, and 271 bp PCR primer sets, resulting in 34.0%, 51.3%, and 79.6% prevalence of the babA2 gene, respectively. The babA2 status determined using the 832 bp and 139 bp PCR primer sets significantly correlated with bacterial density and activity of inflammation, whereas no such correlations were found using the 271 bp PCR primer set. The 139 and 832 bp PCR primer sets concordantly detected the babA2 gene in 93 cases; however, in comparison to the 832 bp PCR primer set, the 139 bp PCR primer set detected additional 50 babA2 cases, whereas only two 832 bp positive cases were missed. The 271 bp PCR primer set missed 32 babA2 cases that were 832 bp and/or 139 bp PCR positive, but tested solely positive in 109 cases. Interestingly, cloning of a subset of 271 bp PCR positive samples revealed amplification of the babA/B gene chimera. Hence, in our opinion, the 271 bp PCR protocol is not a reliable diagnostic tool for detecting the babA2 gene in children. Our results reaffirm previous observations that the use of certain babA2 PCR primer sets can significantly impact estimation of the prevalence and clinical relevance of the H. pylori babA2 gene in children, suggesting babA2 detection methods should be carefully selected.

Toll-like Receptor 9 Agonists in HPV Vaccine Gardasil9

Gardasil9 is a recombinant human papillomavirus (HPV) 9-valent vaccine, containing purified major capsid L1 protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 re-assembled into virus-like particles (VLPs) as the active ingredients. Since the antigens are purified recombinant proteins, in theory Gardasil9 needs a potent adjuvant to generate high and sustained levels of antibodies. Historically, amorphous aluminum hydroxyphosphate sulfate (AAHS), listed as the adjuvant for Gardasil9, was known to require one or more Toll-like receptor agonists, such as the phospholipids in the recombinant hepatitis B vaccine, Recombivax HB®. However, there are no phospholipids in the purified HPV L1 proteins or in Gardasil9. But the Food and Drug Administration (FDA) reports that Gardasil4 does contain recombinant HPV L1-specific DNA fragments, and they may serve as Toll-like receptor 9 agonists in Gardasil9. The author has tested 5 samples of Gardasil9 from 4 manufacturing lots by PCR amplification with a set of degenerate primers followed by heminested PCR or by another 5 sets of non-degenerate nested PCR primers in an attempt to detect all 9 vaccine-relevant HPV type-specific L1 gene DNAs bound to AAHS in the vaccine. Sanger sequencing confirmed the presence of HPV 18, 11, 16 and 6 L1 gene DNA bound to insoluble AAHS nanoparticles, but they were unevenly distributed even within the same vaccine sample. Also, these fragments were at least partially in non-B conformations. Since no L1 gene DNA of HPV 31, 33, 45, 52, and 58 was amplified by the commonly used degenerate PCR primers, the results suggest that these may all be in non-B conformations or may have been removed as contaminants by a purification protocol. Further research is warranted to standardize the HPV DNA fragments in Gardasil which are known to be potent Toll-like receptor 9 agonists.

Novel PCR primers for specific detection of C1, C2 and C3 enterotoxin genes in Staphylococcus aureus

Staphylococcus aureus is the major pathogen that causes clinical infection as well as food poisoning. Enterotoxin produced by Staphylococcus aureus strains are, among others, staphylococcal enterotoxin (SE) A, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEK, SEL and SEM. For SEC, there are three major antigentically distinct SEC subtypes, i.e. SEC1, SEC2 and SEC3, in addition to other molecular variants. The nucleotide sequence homology between SEC1, SEC2, and SEC3 genes is higher than 97%; however, we were able to develop a second set of PCR primers that allowed us to differentiate the three SECs, i.e., SEC1, SEC2 and SEC3, S. aureus strains. These PCR primers and their combinations were C12F/C1BR, C12F/C23R and C3BF/C23R, for specific detection of the SEC1, SEC2 and SEC3 genes of S. aureus strains, respectively. Using these primers, we examined 39 SEC S. aureus isolates, which were obtained from foods that were likely the source of food-borne outbreaks between 1995 and 1997 in central Taiwan, and from the patients of the outbreaks. The results were consistent with those obtained from the first set of primers reported earlier, indicating that the present primers could be used for specific detection of these three toxin types.

Food poisoning caused by sunfish Masturus lanceolatus in Taiwan

Attempts was made to clarify the toxicity and species of causative sunfish fillet concerning with a food poisoning. The frozen causative sunfish fillet was collected to determine its toxicity after two people eating it found poisoned. Tetrodotoxin (TTX) was not detected, however, the butanol extract of remained causative muscle showed ichthyotoxicity to gold carp and caused human, rat and tilapia red blood cells haemolysis. Patients and rats showed some symptoms similar to those caused by palytoxin. Hence, it is possible that this food poisoning is caused by a palytoxin-like compound. Moreover, fish species of the sunfish fillet was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A PCR primer set (L14735/H15149) was used to amplify the partial fragment of the cytochrome b gene. Endonucleases Stu I was used in RFLP. The species of the causative sample was identified as Masturus lamceolatus.

Improved methods for detecting Xylella fastidiosa in pecan and related Carya species

Pecan bacterial leaf scorch (PBLS), caused by Xylella fastidiosa has been reported in Georgia, Louisiana, Texas, New Mexico, Arizona, and California. Accurate methods are critical for the early detection of X. fastidiosa, but the validation of current diagnostic tools for pecan has yet to be investigated. Collected petioles, leaflets, and shoots from pecan and other Carya relatives in Texas, Indiana, and Georgia were used as tissue samples, and to isolate crude xylem sap and gDNA for side-by-side testing using immunological (ELISA) and molecular-based assays [traditional PCR and real-time quantitative PCR (qPCR)]. Isolated crude sap was found to be the most reliable template for ELISA diagnostics. X. fastidiosa-specific genes were amplified with previously published PCR primer sets; however, they revealed non-specific binding. New Xylella-specific primers were subsequently generated and validated using infected tissue from pecan and related Carya species. Two new primer sets (NMU3 and 5) produced expected amplicons specific to X. fastidiosa but did not amplify any non-specific bands of the pecan gDNA. When compared to that of total gDNA as the template in PCR reactions, diluted crude sap was found to be an efficient way to detect X. fastidiosa in pecan petioles. A novel TaqMan qPCR assay was also developed for the detection of X. fastidiosa. The results of the qPCR experiments were equivalent to the traditional PCR amplification when crude sap was used as the template. Comparative PCR analysis confirms that the PCR protocol outlined in this study can be replicated across different laboratories.

Specific detection and quantification of Ralstonia pseudosolanacearum race 4 strains from Zingiberaceae plant cultivation soil by MPN-PCR

The most probable number-polymerase chain reaction (MPN-PCR) method was assessed for the specific detection and quantification of Ralstonia pseudosolanacearum race 4 strains in soil from Zingiberaceae fields in Japan. Based on the genome sequence analysis of two bacterial strains [MAFF 211479 (type I) and MAFF 211472 (type II)] isolated from ginger, race 4 type I- and type II-specific nested PCR primer sets were designed, respectively. The MPN-PCR using these primer sets efficiently detected and quantified the pathogen in naturally and artificially infested soils.

Estimation of trophic niches in myrmecophagous spider predators

Among spiders, taxonomically the most diversified group of terrestrial predators, only a few species are stenophagous and feed on ants. The levels of stenophagy and ant-specialisation vary among such species. To investigate whether stenophagy is only a result of a local specialisation both fundamental and realised trophic niches need to be estimated. Here we investigated trophic niches in three closely-related spider species from the family Gnaphosidae (Callilepis nocturna, C. schuszteri, Nomisia exornata) with different levels of myrmecophagy. Acceptance experiments were used to estimate fundamental trophic niches and molecular methods to estimate realised trophic niches. For the latter two PCR primer sets were used as these can affect the niche breadth estimates. The general invertebrate ZBJ primers were not appropriate for detecting ant DNA as they revealed very few prey types, therefore ant-specific primers were used. The cut-off threshold for erroneous MOTUs was identified as 0.005% of the total number of valid sequences, at individual predator level it was 0.05%. The fundamental trophic niche of Callilepis species included mainly ants, while that of N. exornata included many different prey types. The realised trophic niche in Callilepis species was similar to its fundamental niche but in N. exornata the fundamental niche was wider than realised niche. The results show that Callilepis species are ant-eating (specialised) stenophagous predators, catching mainly Formicinae ants, while N. exornata is an ant-eating euryphagous predator catching mainly Myrmicinae ants.

Identification of Heterodera glycines (Tylenchida; Heteroderidae) Using qPCR.

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea’s soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R2 = 0.996, slope = −3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

Using environmental DNA metabarcoding to map invasive and native invertebrates in two Great Lakes tributaries

AbstractBackgroundAquatic invasive species (AIS) threaten ecosystems and native species.MethodsTo determine spatial distributions of at‐risk native taxa and AIS in two biologically diverse Laurentian Great Lakes tributaries, we extracted environmental DNA (eDNA) from water samples and used a universal PCR primer set targeting the CO1 gene for metabarcoding of selected taxa. We sampled 43 sites for eDNA in each of the Grand and Sydenham rivers in southwestern Ontario.ResultsWe assigned sequences to 49 taxa at the species level and four mollusks to genus level. Detected AIS included two oligochaete worms (Branchiura sowerbyi,Potamothrix moldaviensis), a freshwater jellyfish (Craspedacusta sowerbyi), a calanoid copepod (Skistodiaptomus pallidus), and a bivalve dreissenid mussel (Dreissena rostriformis bugensis). All but D. r. bugensis were previously unreported in these tributaries. Detected native mollusks included one globally endangered species the rayed bean (Villosa fabalis), one provincially listed threatened species the maple leaf mussel (Quadrula quadrula), and several other at‐risk and unique mollusk species of special interest in Ontario, Canada, and the United States (e.g.,Sphaerium fabale,Pyganodon grandis). At several sampling sites in each river, AIS eDNA overlapped with or was near to sites with detections of at‐risk native mollusks. Most AIS and some native taxa demonstrated clustered detection patterns within each river. However, in some cases, independent detections of individual species occurred at individual sites within each river that were relatively far apart. Our findings should be interpreted with some caution due to the limitations of the aquatic “universal” primer set and the availability of comprehensive reference sequence databases.ConclusionResults from eDNA metabarcoding in our study helped reveal invertebrate AIS and at‐risk species distributions and will help direct approaches for conserving biodiversity in each of these Great Lakes tributaries.

Occurrence and frequency of spot form and net form of net blotch disease of barley in Algeria

In Algeria, barley (Hordeum vulgare L.) is the second most cultivated cereal after wheat; however, this crop is challenged by several foliar diseases. Net blotch is one of the most common diseases of barley in Algeria and is caused by the fungus Pyrenophora teres (Died.) Drechsler, which occurs in two forms: P. teres f. teres (Ptt), causing the net form (NFNB), and P.teres f. maculata (Ptm), causing the spot form (SFNB). Since the two forms are morphologically similar but genetically distinct, several PCR primer sets have been developed for their differentiation in the past. In this study, net blotch symptom types were monitored in 58 fields, through almost all cereal-growing areas in Algeria, then 212 P. teres mono-spore isolates were analyzed with respect to the occurrence of Ptt and Ptm by using two type-specific primer pairs. The results indicate that Ptt is the dominating type of net blotch in Algeria and was prevalent in almost all provinces surveyed, while Ptm was found less frequently. This is the first characterization of the Algerian net blotch population with the aim to distinguish both forms and, therefore, could contribute to a wider knowledge of the epidemics of this important plant pathogen.

Development of a multiplex PCR test-system for detection of BHV-1, BVDV, Chlamydia spp. and Mycoplasma spp.

This study describes development of a multiplex PCR assay for detection of BHV-1, BVDV, Chlamydia spp. and Mycoplasma spp. infections in bovines. The assay was developed using genomic DNA and RNA and four sets of PCR primers targeting 16S rRNA genes of Chlamydia spp., Mycoplasma spp., 5’-UTR of Bovine viral diarrhea virus, gE of Bovine herpesvirus-1, respectively. A total of 100 tissue samples were collected from cattle suspected to be infected with the viral and bacterial pathogens (BVDV, BHV-1, Chlamydia spp. and Mycoplasma spp.) from different regions of Ukraine. A part of sample was stored at –50°C for isolation of genomic DNA and RNA. The multiplex PCR assay was optimized in the study. The specific primers designed and used in the study were found sensitive and specific in amplifying target genes viz. 16S rRNA, gE, 5’-UTR of Chlamydia spp., Mycoplasma spp., BHV-1 and BVDV, respectively. The PCR primers used in the optimization of multiplex PCR assay for detection of Bovine viral diarrhea virus, Bovine herpesvirus-1, Chlamydia spp., Mycolasma spp. could amplify 221 bp, 111 bp, 386 bp, 279 bp products, respectively. Non specific amplification was not observed

Papillomavirus DNA is not Amplifiable from Bladder, Lung, or Mammary Gland Cancers in Dogs or Cats.

Simple SummaryPapillomaviruses (PVs) are well established to cause genital and oral cancer in humans. Additionally, some evidence suggests that these viruses may also have a role in the development of human lung, breast, and bladder cancer. Recent studies have revealed that PVs can influence the development of some cancers in cats and, less frequently, in dogs. In the present study, 47 lung, mammary gland, and bladder cancers in dogs and 25 cancers in cats were evaluated for evidence of any role PVs may have in cancer development. Histology did not reveal any lesions suggestive of PV infection, and no PV DNA was amplified from any cancer. Therefore, these findings suggest that PVs do not frequently infect these tissues and are therefore unlikely to be significant factors in the development of lung, mammary gland, or bladder cancer in either dogs or cats.Papillomaviruses (PVs) cause around 5% of all human cancers, including most cervical cancers and around a quarter of all oral cancers. Additionally, some studies have suggested that PVs could cause a proportion of human lung, breast, and bladder cancers. As PVs have been associated with skin cancer in cats and, more rarely, dogs, it was hypothesized that these viruses could also contribute to epithelial cancers of the lung, mammary gland, and bladder of dogs and cats. Formalin-fixed paraffin-embedded samples of 47 canine and 25 feline cancers were examined histologically for evidence of PV infection. Additionally, three sets of consensus PCR primers were used to amplify PV DNA from the samples. No histological evidence of PV infection was visible in any of the cancers. DNA from a bovine PV type was amplified from one sample, while two different samples were found to contain human PV DNA. However, these were considered to be contaminants, and no canine or feline PV types were amplified from any of the cancers. These results suggest that PVs do not frequently infect the lung, mammary gland, or bladder of dogs and cats and therefore are unlikely to be significant factors in the development of cancers in these tissues.

Detection and first molecular characterisation of three picornaviruses from diarrhoeic calves in Turkey.

The involvement of picornaviruses in calf diarrhoea was evaluated by the analysis of 127 faecal samples collected from diarrhoeic calves during 2014-2016. Virus detections were carried out by PCR using generic or specific primer pairs. One-third of the faecal samples (33.86%) were found to be positive for one or more of the studied viruses. Bovine kobuvirus was detected in 22.83%, bovine hungarovirus in 11.02%, while bovine enterovirus 1 in 5.51% of the samples. The sequences of the PCR products indicated the existence of novel variants in all the three virus species. When comparing the partial sequences, the nucleotide sequence identities between our newly detected viruses and those previously deposited to the GenBank ranged between 76 and 99%. Phylogenetic analyses revealed a novel lineage within the species Hunnivirus A. Our findings suggest that these viruses should be regarded as possible aetiological agents of calf diarrhoea. Based on the newly determined sequences, we designed and tested a new generic PCR primer set for the more reliable detection of bovine hungaroviruses. This is the first report on the molecular detection of the presence of bovine hungarovirus, bovine kobuvirus and bovine enterovirus 1 in the faecal samples of diarrhoeic calves in Turkey.

Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method.

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.

TaqMan real-time and conventional nested PCR tests specific to yellow head virus genotype 7 (YHV7) identified in giant tiger shrimp in Australia

In 2013, a unique seventh yellow head virus genotype (YHV7) was detected in Black Tiger shrimp (Penaeus monodon) broodstock that suffered high mortality following their capture from Joseph Bonaparte Gulf (JBG) in northern Australia. To assist with its diagnosis and assessment of its distribution, prevalence and pathogenicity, YHV7-specific TaqMan real-time qPCR and conventional nested PCR primer sets were designed to ORF1b gene sequences divergent from the other YHV genotypes. Using high (≥108) copies of plasmid (p)DNA controls containing ORF1b gene inserts of representative strains of YHV genotypes 1–7, both PCR tests displayed specificity for YHV7. Amplifications of serial 10-fold dilutions of quantified YHV7 pDNA and synthetic ssRNA showed that both tests could reliably detect 10 genome copies. Pleopods/gills from wild P. monodon sourced from locations in geographically disparate regions across northern Australia as well as 96 juveniles (48 either appearing normal or displaying signs of morbidity) from a commercial pond experiencing mortalities were screened to partially validate the diagnostic capacity of the qPCR test. Based on these data and PCR primer/probe sequence mismatches with other newly identified YHV genotypes, both YHV7-specific PCR tests should prove useful in the sensitive detection and discrimination of this genotype from YHV 2 (gill-associated virus) and YHV6 that can occur in Australian P. monodon, as well as from YHV genotypes currently listed as exotic to Australia.

Foodborne Illness Case Detected <i>Unicapsula seriolae</i> by PCR-Based Test to Detect A Wide Range of 18S Ribosomal RNA Gene of Genus <i>Kudoa</i>

In April 2018 in Toyama Prefecture, cases of foodborne illnesses possibly caused by Kudoa septempunctata were reported from eating raw flounder, amberjack, and tuna sashimi. Leftover foods and feces from two of the patients were immediately tested for K. septempunctata, but there were none detected. Therefore, we suspected the presence of myxosporean other than K. septempunctata. We performed a PCR-based test with specific primers, developed by us, to detect a wide range of 18S ribosomal DNA (rDNA) from the genus Kudoa and Unicapsula. In one of the leftover foods, amberjack sashimi, we detected Unicapsula seriolae (U. seriolae) 18S rDNA using another set of specific primers. Two rounds of PCR amplification were carried out on DNA from stool samples of the patients using both primer sets to detect 18S rDNA of the genus Kudoa or of U. seriolae. Lastly, 18S rDNA of U. seriolae was also detected in feces from the two patients by sequence analysis of the PCR products. Therefore, U. seriolae contamination was predicted to be responsible for illness in the reported cases. Additionally in this study, we were able to demonstrate the use of our PCR primer sets to screen for the genus Kudoa and Unicapsula.

Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39

Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 105 CFU/ml (4.5 ng/μl ADN) or 102 CFU/ml (0.88 ng/μl DNA) or in a minimal concentration of 0.098 ng/μl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers.

Community dynamics of Neocallimastigomycetes in the rumen of yak feeding on wheat straw revealed by different primer sets

Abstract Anaerobic fungi (Neocallimastigomycetes) play an important role in fermenting lignin-rich plant biomass into sugars in the rumen of animals, representing a very promising enzyme resource to contribute to the conversion of plant biomass into biofuels. However, current studies about their functions mainly focus on limited species, and little is known about the coordination of different members of the anaerobic fungi in the digestion process of plant fibres. In this study, the community composition of anaerobic fungi in the rumens of yaks at five different time points (1, 3, 5, 7.5 and 24 h after feeding wheat straw) was investigated employing a cultivation-independent method using ITS clone libraries. Comparison of five pairs of primers showed that PCR primer sets could have clear amplification bias and therefore potentially affect the interpretation of the resulting fungal community structure; then two primer sets GM1/MNGM2 and ITS1/ITS4 were selected. Among the 398 sequences from 10 clone libraries, 18 operational taxonomic units (OTUs) of Neocallimastigomycetes were obtained, covering five known genera and one yet uncultured lineage. OTUs belonging to the bulbous-type morphotype (Caecomyces- or Cyllamyces-related) and the rhizoidal genus Neocallimastix were abundant and predominantly present, representing 62.7% and 19.3% OTUs respectively. In all the later samples taken from 3 h to 24 Neocallimastigomycetes h after feeding, a relatively stable community composition was revealed: members of Neocallimastix increased to represent 43.4–49.4% and the bulbous-type morphotype declined to represent 39.5–42.7%. This implies a substantial turnover and synergy between bulbous and rhizoidal morphotypes of anaerobic fungi during the process of fibre digestion. Our study provided the first insight into the in vivo temporal change in the anaerobic fungal community, and the role of Neocallimastigomycetes with a bulbous morphotype in the degradation of plant cell wall in the yak rumen.

Development of a new set of PCR primers for eDNA metabarcoding decapod crustaceans

The Decapoda is one of the largest orders within the class Malacostraca, comprising approximately 14,000 extant species and including many commercially important species. For biodiversity monitoring in a non-invasive manner, a new set of PCR primers was developed for metabarcoding environmental DNA (eDNA) from decapod crustaceans. The new primers (herein named “MiDeca”) were designed for two conservative regions of the mitochondrial 16S rRNA gene, which amplify a short, hyper-variable region (153–184 bp, 164 bp on average) with sufficient interspecific variations. With the use of MiDeca primers and tissue-derived DNA extracts, we successfully determined those sequences (154–189 bp) from 250 species, placed in 186 genera and 65 families across the suborder Dendrobranchiata and 10 of the 11 infraorders of the suborder Pleocyemata. We also preliminarily attempted eDNA metabarcoding from natural seawater collected at Banda, Tateyama, the Pacific coast of central Japan and detected 42 decapod species including 34 and 8 species with sequence identities of > 98% and 80–98%, respectively. The results suggest the usefulness of eDNA metabarcoding with MiDeca primers for biodiversity monitoring of the decapod species. It appears, however, that further optimisation of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts.