Detection of Rhopalosiphum insertum (apple-grass aphid) predation by the predatory mite Anystis baccarum using molecular gut analysis

Abstract 1 A simple, yet sensitive polymerase chain reaction based technique was developed for the detection of the apple‐grass aphid Rhopalosiphum insertum in the gut of Anystis baccarum, a predatory mite. 2 A range of conserved polymerase chain reaction primers for insect mitochondrial and ribosomal DNA were tested in order to amplify R. insertum DNA. The mitochondrial DNA primers LrRNAR2 + N1F1, amplified a region between the ND1 and large subunit RNA genes. 3 DNA sequencing of the R. insertum ND1‐LRNA polymerase chain reaction product allowed aphid‐specific polymerase chain reaction primers to be designed. These amplified a 283‐bp product from individual aphids. No polymerase chain reaction product was amplified from individual A. baccarum. 4 Using the aphid‐specific primers against A. baccarum fed on R. insertum, the diagnostic 283‐bp product was amplified. 5 Two restriction enzymes (RsaI and AluI) produced patterns that allowed unambiguous identification of R. insertum DNA from that of Macrosiphum euphorbiae and Myzus persicae.     

Identification of Aeromonas schubertii and Aeromonas jandaei by using a polymerase chain reaction-probe test

Abstract Two oligonucleotide primers were used in a polymerase chain reaction-protocol to amplify a region (approx. 850 bp) of the 16S rRNA gene of Aeromonas schubertii and Aeromonas jandaei . Hybridization of the polymerase chain reaction products to specific internal probes provided a highly specific method for the identification of these two species.     

Development of primers to characterize the mitochondrial control region of the snow leopard (Uncia uncia)

The snow leopard (Uncia uncia) is a rare carnivore living above the snow line in central Asia. Using universal primers for the mitochondrial genome control region hypervariable region 1 (HVR1), we isolated a 411‐bp fragment of HVR1 and then designed specific primers near each end of this sequence in the conserved regions. These primers were shown to yield good polymerase chain reaction products and to be species specific. Of the 12 snow leopards studied, there were 11 segregating sites and six haplotypes. An identification case of snow leopard carcass (confiscated by the police) proved the primers to be a useful tool for forensic diagnosis in field and population genetics studies.     

Short microsatellite DNA markers for the red fox (Vulpes vulpes)

Seven short microsatellite loci (< 165 bp) were characterized for red foxes for the amplification of degraded DNA extracted from historical samples. Following polymerase chain reaction (PCR) using primers developed in the domestic dog, red fox‐specific primers were designed within the flanking regions. The number of detected alleles ranged between six and 15 alleles and the expected heterozygosities ranged between 0.67 and 0.92. No deviations from Hardy–Weinberg equilibrium were detected for any of the markers.     

Bi‐parentally inherited species‐specific markers identify hybridization between rainbow trout and cutthroat trout subspecies

Eight polymerase chain reaction primer sets amplifying bi‐parentally inherited species‐specific markers were developed that differentiate between rainbow trout (Oncorhynchus mykiss) and various cutthroat trout (O. clarki) subspecies. The primers were tested within known F₁ and first generation hybrid backcrosses and were shown to amplify codominantly within hybrids. Heterozygous individuals also amplified a slower migrating band that was a heteroduplex, caused by the annealing of polymerase chain reaction products from both species. These primer sets have numerous advantages for native cutthroat trout conservation including statistical genetic analyses of known crosses and simple hybrid identification.     

Species‐specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

Inteins (internal proteins) are self‐splicing transportable genetic elements present in conserved regions of housekeeping genes. The study highlights the importance of intein as a potential diagnostic marker for species‐specific identification of Candida tropicalis, a rapidly emerging opportunistic human pathogen. Initial steps of primer validation, sequence alignment, phylogenetic tree analysis, gel electrophoresis and real‐time polymerase chain reaction (PCR) assays were performed to confirm the specificity of the designed primers. The primers were selective for C. tropicalis with 100% inclusivity and showed no cross‐species or cross‐genera matches. The established technique is a prototype for developing multifaceted PCR assays and for point‐of‐care testing in near future.

Significance and Impact of the Study

Development of molecular markers for specific detection of microbial pathogens using real‐time polymerase chain reaction (PCR) is an appealing and challenging technique. A real‐time PCR is an emerging technology frequently used to detect the aetiologic agents. In recent times, designing species‐specific primers for pathogen detection is gaining momentum. The method offers rapid, accurate and cost‐effective strategy to identify the target, thus providing sufficient time to instigate appropriate chemotherapy. The study highlights the use of intein DNA sequence as molecular markers for species‐specific identification of Candida tropicalis. The study also offers a prototype model for developing multifaceted PCR assays using intein DNA sequences, and provides a developmental starting point for point‐of‐care testing in near future.     

Characterization of microsatellite loci in the Kaka,Nestor meridionalis

Eight microsatellite loci were characterized from the Kaka (Nestor meridionalis), a New Zealand parrot, using a polymerase chain reaction‐based isolation technique. Locus‐specific primers were used to genotype nine Kaka populations and tested on 25 other species of parrot. The number of alleles observed within Kaka ranged from one to 16 in the 12–126 individuals screened and two loci exhibited greater than 60% heterozygosity. Furthermore, these primers are likely to be useful in population‐level studies of two other New Zealand parrots.     

Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.     

马铃薯S病毒RT-PCR检测技术的研究

从感染PVS病毒的马铃薯病叶组织中提取出病毒的RNA,进行反转录cDNA的合成,用特异性引物进行PCR扩增,得到一条长度约为199 bp的特异性PCR扩增产物,与理论设计的外壳蛋白基因大小一致。在基因水平上为PVS的检测提供了一种快速、灵敏、简便的新方法,为PVS的防治提供了有效手段。     

Identification by polymerase chain reaction (PCR) of Marshallagia marshalli and Ostertagia gruehneri from Svalbard reindeer

A polymerase chain reaction (PCR) assay to identify two common abomasal nematodes Marshallagia marshalli and Ostertagia gruehneri of Svalbard reindeer was developed. Species-specific PCR primers were designed from internal transcribed spacer (ITS)-2 sequences of rDNA and validated using morphologically identified adult male and female nematodes. Using the species-specific primers, a 110 bp fragment was amplified from M. marshalli and its minor morph Marshallagia occidentalis and a 149 bp fragment was amplified from Ostertagia gruehneri and its minor morph Ostertagia arctica. No PCR products were amplified from the third rare species, Teladorsagia circumcincta, or DNA from the reindeer host. The assay provides a useful tool to estimate species composition for both sexes in this nematode community.     

Complete mitochondrial genome of the black-headed snake Sibynophis collaris (Squamata, Serpentes, Colubridae)

The black-headed snake Sibynophis collaris (Reptilia, Squamata, Colubridae) is a least concern species in the world. Two universal and two specific polymerase chain reaction (PCR) primers were used for long PCRs to amplify the whole mitochondrial genome of S. collaris. The products were subjected to do sequencing reactions. The complete genome is 17,163 bp in size, containing 37 genes coding for 13 proteins, 2 rRNAs, 22 tRNAs, and 2 control regions (CRI and CRII). The results could play an important role in the preservation of genetic resources for helping conservation of the endangered species.     

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.     

Gel‐free species identification using melt‐curve analysis

DNA‐based identification of organisms is an important tool in biosecurity, ecological monitoring and wildlife forensics. Current methods usually involve post‐polymerase chain reaction (PCR) manipulations (e.g. restriction digest, gel electrophoresis), which add to the expense and time required for processing samples, and may introduce error. We developed a method of species identification that uses species‐specific primers and melt‐curve analysis, and avoids post‐PCR manipulation of samples. The method was highly accurate when trialled on DNA from six large carnivore species from Tasmania, Australia. Because of its flexibility and cost‐effectiveness, this method should find wide use in many areas of applied biological science.     

Simultaneous identification of Grapholita molesta Busck and Grapholita dimorpha Komai by PCR‐RFLP

Although several molecular diagnostic techniques are available for the identification of the apple‐feeding pests Grapholita molesta Busck and Grapholita dimorpha Komai, these pests are severely affecting apple orchards in Korea. These two pests may be misidentified or the available molecular diagnostic techniques may not facilitate the simultaneous identification of the morphological features of both species. In this study, we developed a multiplex assay for these two species using the polymerase chain reaction – restriction fragment length polymorphism (PCR‐RFLP) method. Sixty‐two specimens were collected from apples presumed infested with moth larvae and from pheromone traps from 2013 to 2014. Both species were identified morphologically, and a partial region of the cytochrome b gene was sequenced to design primers for PCR‐RFLP. Digestion profiles of G. molesta and G. dimorpha, using the Sau3A1 restriction enzyme, were characterized using three DNA fragments each for G. molesta (363 bp, 91 bp and 31 bp) and G. dimorpha (220 bp, 234 bp and 31 bp). The RFLP assay developed for both species in this study was more efficient and accurate than other currently used diagnostic assays and would be helpful to identify field‐collected specimens for pest control research.     

DNA probe and PCR-specific reaction for Lactobacillus plantarum

A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus , albeit more weakly. Two internal primers of this probe were selected (LbPl1 and LbPl2) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum .     

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was developed to detect and identify typical Leuconostoc species. This method utilises a set of specific primers for amplification of the 16S rDNA region of typical Leuconostoc species. All Leuconostoc-type strains, all Leuconostoc isolates from kimchi, Korea's traditional, fermented vegetable product, and strains from closely related genera were examined to verify the identification by this method. The primers resulted in amplification only for nine typical Leuconostoc spp., but not for any other genera tested. The size of the amplified products was 976 bp and the amplicons of the different species could be differentiated from each other with MseI, HaeIII and Tsp509I endonucleases, except for the species Leuconostoc argentinum and Leuconostoc lactis, which were indistinguishable. A PCR-RFLP method for the typical Leuconostoc species was optimized to identify a large number of isolates from fermented vegetable product. This PCR-RFLP method enables the rapid and reliable identification of Leuconostoc species and to distinguish them from the other phylogenetically related lactic acid bacteria in food samples.     

Fecal DNA analysis for identifying species and sex of sympatric carnivores: a noninvasive method for conservation on the Tsushima Islands, Japan

Fecal analysis is a useful tool for the investigation of food habits and species identity in mammals. However, it is generally difficult to identify the species based on the morphological features and contents of feces deposited by mammals of similar body size. Therefore we developed noninvasive DNA analysis methods using fecal samples for identification of the species and sex of four small sympatric carnivores living on the Tsushima Islands of Japan: the leopard cat (Felis bengalensis), Japanese marten (Martes melampus), Siberian weasel (Mustela sibirica), and feral cat (Felis catus). Based on DNA sequence data from previous phylogenetic studies, we designed species-specific primers for polymerase chain reaction (PCR) amplification of the partial mitochondrial cytochrome b gene (112-347 bp) to identify the species and primers for the partial SRY gene (135 bp) to determine the sex. Due to the adjustment of PCR conditions, those specific DNA fragments were successfully amplified and then applied for species and sex identification. Nucleotide sequences obtained from the PCR products corresponded with cytochrome b sequences of the carnivore species expected. The protocol developed could be a valuable tool in the management and conservation of the four carnivore species occurring on the Tsushima Islands.     

Wide distribution of short interspersed elements among eukaryotic genomes.

Most short interspersed elements (SINEs) in eukaryotic genomes originate from tRNA and have internal promoters for RNA polymerase III. The promoter contains two boxes (A and B) spaced by approximately 33 bp. We used oligonucleotide primers specific to these boxes to detect SINEs in the genomic DNA by polymerase chain reaction (PCR). Appropriate DNA fragments were revealed by PCR in 30 out of 35 eukaryotic species suggesting the wide distribution of SINEs. The PCR products were used for hybridization screening of genomic libraries which resulted in identification of four novel SINE families. The application of this approach is illustrated by discovery of a SINE family in the genome of the bat Myotis daubentoni. Members of this SINE family termed VES have an additional B-like box, a putative polyadenylation signal and RNA polymerase III terminator.