scholarly journals PCR primers comparisons for a successful Tuber spp. DNA region amplification in routine identifications / Primerjava PCR začetnih oligonukleotidov za uspešno pomnoževanje DNA regije Tuber spp. pri rutinski identifikaciji

2020 ◽  
Vol 61 (2) ◽  
pp. 229-238
Author(s):  
Tina Unuk Nahberger ◽  
Hojka Kraigher ◽  
Tine Grebenc
Keyword(s):  
Pcr Amplification ◽  
Its Region ◽  
Pcr Primers ◽  
Target Sequence ◽  
Tuber Aestivum ◽  
Root Tips ◽  
Late 20Th Century ◽  
Amplification Success ◽  
Tuber Species ◽  
Tuber Spp

Since late 20th century DNA sequencing became the method of choice method in precision species identification. The ITS region is one of the official fungal barcoding DNA markers, although in some cases sequencing of the ITS region may, due to misidentification, mislabeling or nomenclature errors in public databases, lead to incorrect or insufficient identification, as is currently a case in the genus Tuber. The aim of this study was to test, which ITS primer pairs are most appropriate and optimal for Tuber species DNA region amplification. Thereby we (1) compared amplification success for different Tuber species using fungal specific primer pair ITS1f and ITS4 and (2) compared amplification success using different ITS primer pair combinations in amplifying DNA region an example species Tuber aestivum. Based on results, Tuber aestivum was one of the most reluctant Tuber species in this study and in most cases failed to amplify with the above primer pair. After comparing different ITS primer pairs, we conclude that the primer pair ITS5 and ITS7 is the most appropriate primer pair for amplification DNA region of T. aestivum as it resulted in high amplification success from ectomycorrhizal root tips. Based on sequences, gained from public databases, we found that ITS1f and ITS6 primers have a mismatch in one base pair compared to the target sequence of Tuber aestivum, thus resulting in poor or no amplification success. Although primer pair ITS5 and ITS7 in our study was proven to be the most appropriate primer pair in amplifying DNA region Tuber aestivum species, further analysis about appropriateness of it for a general barcoding and identification of ectomycorrhiza in complex community samples is needed.  Keywords: Tuber spp., ITS region, PCR amplification, ITS primers    Izvleček Od konca 20. stoletja je določanje nukleotidnega zaporedja DNA postalo ena izmed pogosteje uporabljenih metod za določanje vrst. ITS regija je edna izmed uradnih glivnih DNA markerjev, čeprav lahko določanje nukleotidnega zaporedja le-te, v nekaterih primerih, predvsem zaradi napačne določitve, označevanja oziroma napak v nomenklaturi v javnih bazah podatkov, privede do napačne oziroma nenatančne določitve vrst, kar je trenutno težava pri določitvi vrst iz rodu Tuber. Namen te študije je bil testirati kateri pari ITS začetnih oligonukleotidov so najbolj primerni in optimalni za pomnoževanje DNA regij gliv iz rodu Tuber. S tem namenom smo v študiji (1) primerjali uspešnost pomnoževanja DNA regije različnih vrst iz rodu Tuber, z uporabo glivno specifičnih začetnih oligonukleotidov ITS1f in ITS4 ter hkrati (2) primerjali uspešnost pomnoževanja DNA regije vrste Tuber aestivum z uporabo različnih ITS začetnih oligonukleotidov. Na podlagi rezultatov ugotavljamo, da je vrsta T. aestivum izmed vseh analiziranih gliv iz rodu Tuber, bila najtežavnejša vrsta v naši študiji, saj je v večini primerov pomnoževanje DNA regije te vrste z uporabo glivno specifičnih začetnih oligonukleotidov ITS1f in ITS4 bilo neuspešno. Po primerjavi uspešnosti pomnoževanja z različnimi ITS začetnimi oligonukelotidi ugotavljamo, da sta bila v naši študiji ITS začetna oligonukleotida ITS5 in ITS7 najprimernejša za pomnoževanje DNA regije vrste T. aestivum, saj je bila uspešnost pomnoževanja iz ektomikoriznih vršičkov v tem primeru največja. Na podlagi T. aestivum nukleotidnih zaporedij pridobljenih iz javnih podatkovnih baz ugotavljamo, da je za začetna oligonukleotida ITS1f in ITS6 značilno neujemanje s tarčnim nukleotidnim zaporedjem (T. aestivum) v enem baznem paru, kar se lahko odraža bodisi v slabšem pomnoževalnem uspehu ali v nepomnoževanju na splošno. Kljub temu, da v naši študiji ugotavljamo, da sta začetna oligonukleotida ITS5 in ITS7 najprimernejša za pomnoževanje DNA regije glive T. aestivum, so potrebne nadaljnje analize, s katerimi bi potrdili splošno primernost omenjenega para ITS5/ITS7 za pomnoževanje DNA regije ne samo vrst iz rodu Tuber, temveč za določanje ektomikoriznih glivnih združb na splošno.   Ključne besede: Tuber spp., ITS regija, PCR pomno­ževanje, ITS začetni oligonukleotidi

scholarly journals Genome size and microsatellites: the effect of nuclear size on amplification potential

Genome ◽  
2002 ◽  
Vol 45 (1) ◽  
pp. 212-215 ◽  
Author(s):  
Trenton WJ Garner
Keyword(s):  
Genome Size ◽  
Microsatellite Dna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Amplification Success ◽  
Pcr Products ◽  
Negative Effect ◽  
C Value ◽  
Primer Sets ◽  
Using Data

Although the frequency of microsatellite DNA regions generally increases with increasing genome size, genome size has a negative effect on polymerase chain reaction (PCR) amplification. Thus, researchers developing sets of PCR primers, as is commonly done for microsatellite DNA regions, may encounter greater difficulty when working with species that have larger genomes. I investigated the effect of genome size on overall amplification success using data from nine different metazoan taxa. The proportion of primer sets that did not amplify PCR products was strongly and positively correlated with the haploid C value of the target species. Increasing genome size may affect amplification success negatively because of a decrease in target:nontarget DNA or by dilution of the available primer pool by nonspecific binding.Key words: microsatellites, genome size, amplification success.

scholarly journals First Report of Phytophthora ramorum on Coast Redwood in California

Plant Disease ◽  
2002 ◽  
Vol 86 (11) ◽  
pp. 1274-1274 ◽  
Author(s):  
P. E. Maloney ◽  
D. M. Rizzo ◽  
S. T. Koike ◽  
T. Y. Harnik ◽  
M. Garbelotto
Keyword(s):  
Pcr Amplification ◽  
Its Region ◽  
Pcr Primers ◽  
Phytophthora Ramorum ◽  
Natural Forests ◽  
Coast Redwood ◽  
Specific Pcr ◽  
Rdna Sequences ◽  
Quercus Spp ◽  
The Impact

Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from discolored leaves and cankers on small branches (<0.5 cm in diameter) on 27 coast redwood (Sequoia sempervirens) saplings (2 to17 cm in diameter) at two locations in California (Jack London State Park, Sonoma County and Henry Cowell State Park, Santa Cruz County). Symptoms were observed on branches throughout the crowns of affected trees. Isolates were identified as P. ramorum by their abundant chlamydospores and caducous, semi-papillate sporangia (2) and internal transcribed spacer (ITS) rDNA sequences identical to those of P. ramorum from Quercus spp., Lithocarpus densiflorus, and Rhododendron (1,2). P. ramorum was also detected in dying basal sprouts on mature redwood trees from an additional five locations in coastal California by polymerase chain reaction (PCR) amplification of the ITS region using DNA extracted from symptomatic tissue and P. ramorum-specific PCR primers. To test for pathogenicity, foliage inoculations were conducted on redwood seedlings in two trials by misting 30 leaves per trial (five leaves per seedling plus controls) with sterile distilled water and then pinning inoculum plugs to the upper surface of leaves. Inoculation resulted in lesions of 1 to 20 mm on individual leaves, and P. ramorum was recovered from 43% of inoculated leaves. Symptoms were not restricted to inoculated leaves because 15 inoculations of individual leaves led to discoloration of two or more adjacent leaves. On one inoculation, 60 mm of the adjacent stem was killed. Stems of redwood seedling (approximately 1 cm in diameter) were wound inoculated (1) in two trials consisting of 10 inoculated seedlings per trial plus 10 controls. After 6 weeks, lesion lengths in the cambium caused by P. ramorum averaged 13.7 mm (range 4 to 21 mm). P. ramorum was recovered from 100% of inoculated stems. Entire branches near the inoculation point became chlorotic even though no direct connection was evident between the lesion and the branches. No chlorosis was observed among the control inoculations. Mean lesion lengths of inoculated stems were significantly greater in both trials than those of control inoculations (mean 6.2 mm) at P < 0.05 based on analysis of variance (ANOVA). Redwood saplings (2.5 to 4.5 cm in diameter) were also wound inoculated in a separate trial. No phloem or cambial discoloration was observed after 7 weeks, but necrotic lesions in the xylem had a mean length of 39 mm (range 12 to 73 mm). In addition, narrow streaks, 1 to 2 mm in diameter, were also noted in the xylem extending from the necrotic areas upward to 90 cm. P. ramorum was recovered from 70% of inoculated stems in this trial. Mean lesion lengths of P. ramorum were significantly greater in all trials than those of control inoculations (mean 20 mm) at P < 0.05 based on ANOVA. While P. ramorum causes a lethal canker on Quercus spp. and L. densiflorus (1), we have not observed unusual mortality or disease symptoms on overstory redwoods in natural forests. The impact of infection by P. ramorum on understory redwoods is also unclear. However, the pathogen appears to be able to kill sprouts. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.

scholarly journals Morpho-molecular identification and first report of Fusarium equiseti in causing chilli wilt from Kashmir (Northern Himalayas)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ammarah Hami ◽  
Rovidha S. Rasool ◽  
Nisar A. Khan ◽  
Sheikh Mansoor ◽  
Mudasir A. Mir ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Pcr Amplification ◽  
Its Region ◽  
Fungal Species ◽  
Pathogenicity Test ◽  
Kashmir Valley ◽  
Fusarium Equiseti ◽  
Infected Root ◽  
Fusarium Sp ◽  
High Degree

AbstractChilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.

scholarly journals Effect of earthworms on mycorrhization, root morphology and biomass of silver fir seedlings inoculated with black summer truffle (Tuber aestivum Vittad.)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tina Unuk Nahberger ◽  
Gian Maria Niccolò Benucci ◽  
Hojka Kraigher ◽  
Tine Grebenc
Keyword(s):  
Root System ◽  
Root Biomass ◽  
Fine Root ◽  
Abies Alba ◽  
Fine Root Biomass ◽  
Root Tip ◽  
Silver Fir ◽  
Tuber Aestivum ◽  
Root Tips ◽  
Root Parameters

AbstractSpecies of the genus Tuber have gained a lot of attention in recent decades due to their aromatic hypogenous fruitbodies, which can bring high prices on the market. The tendency in truffle production is to infect oak, hazel, beech, etc. in greenhouse conditions. We aimed to show whether silver fir (Abies alba Mill.) can be an appropriate host partner for commercial mycorrhization with truffles, and how earthworms in the inoculation substrate would affect the mycorrhization dynamics. Silver fir seedlings inoculated with Tuber. aestivum were analyzed for root system parameters and mycorrhization, how earthworms affect the bare root system, and if mycorrhization parameters change when earthworms are added to the inoculation substrate. Seedlings were analyzed 6 and 12 months after spore inoculation. Mycorrhization with or without earthworms revealed contrasting effects on fine root biomass and morphology of silver fir seedlings. Only a few of the assessed fine root parameters showed statistically significant response, namely higher fine root biomass and fine root tip density in inoculated seedlings without earthworms 6 months after inoculation, lower fine root tip density when earthworms were added, the specific root tip density increased in inoculated seedlings without earthworms 12 months after inoculation, and general negative effect of earthworm on branching density. Silver fir was confirmed as a suitable host partner for commercial mycorrhization with truffles, with 6% and 35% mycorrhization 6 months after inoculation and between 36% and 55% mycorrhization 12 months after inoculation. The effect of earthworms on mycorrhization of silver fir with Tuber aestivum was positive only after 6 months of mycorrhization, while this effect disappeared and turned insignificantly negative after 12 months due to the secondary effect of grazing on ectomycorrhizal root tips.

scholarly journals Quantitative Measure of Small-Subunit rRNA Gene Sequences of the Kingdom Korarchaeota

1998 ◽  
Vol 64 (12) ◽  
pp. 5064-5066 ◽  
Author(s):  
Clifford F. Brunk ◽  
Nicole Eis
Keyword(s):  
Internal Standard ◽  
Pcr Amplification ◽  
Quantitative Measure ◽  
Pcr Primers ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rdna Sequence ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Rdna Sequences

ABSTRACT Comparative PCR amplification of small-subunit (SSU) rRNA gene (rDNA) sequences indicates substantial preferential PCR amplification of pJP27 sequences with korarchaeote-specific PCR primers. The coamplification of a modified SSU rDNA sequence can be used as an internal standard to determine the amount of a specific SSU rDNA sequence.

scholarly journals Identification and Evolutionary Characterization of Papillomavirus Sequences in New World Monkeys (Genera Sapajus and Alouatta) from Argentina

2021 ◽  
Author(s):  
Candelaria Sanchez Fernandez ◽  
Elisa M Bolatti ◽  
Andres C.A. Culasso ◽  
Diego Chouhy ◽  
Martin M Kowalewski ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Pcr Primers ◽  
Host Tree ◽  
New World Monkeys ◽  
Alouatta Caraya ◽  
Captive Populations ◽  
L1 Protein ◽  
Viral Sequences ◽  
Northeastern Argentina

Abstract Objective: In this study, we investigated the occurrence of papillomavirus (PV) infection in non-human primates (NHP, Platyrrhine) of northeastern Argentina by using broad-spectrum PCR primers at the L1 gene. In addition, we conducted a phylogenetic and coalescence analysis of viral sequences to explore their evolutionary history and evaluate the co-speciation hypothesis in the context of primate evolution. Methods: We obtained samples of 57 individuals from wild and captive populations of Alouatta caraya, Sapajus nigritus and Sapajus cay. We assessed PV infection by PCR amplification with the CUT primer system and sequencing of 337 bp (112 amino acids) of the L1 protein. The viral sequences were analyzed by phylogenetic and Bayesian coalescence methods to estimate the age of the most common recent ancestor (tMCRA) with BEAST, v1.4.8 software. We evaluated viral/host tree congruence with TreeMap v3.0. Results: We identified two novel putative PV sequences of the genus Gamma- PV in Sapajus sp and Alouatta caraya (SPV1 and AcPV1, respectively). The tMRCA of SPV1 was estimated at 11,941,682 years before present (ybp) and that of AcPV1 at 46,638,071 ybp, both predating the coalescence times of their hosts: 6.4 million years (MYA) and 6.8 MYA, respectively. Based on the comparison of primate and viral phylogenies, we could not reject the null hypothesis that the PV tree is no more congruent with the host tree than a random tree would be (P>0.05). Thus, a model of virus-host coevolution was rejected. Conclusion: This study presents the first report of PV infection in Platyrrhine species from Argentina, expands the range of described hosts for these viruses, and proposes new scenarios for their origin and dispersal.

scholarly journals Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations

2001 ◽  
Vol 47 (8) ◽  
pp. 1384-1389 ◽  
Author(s):  
Mark S Turner ◽  
Sarah Penning ◽  
Angela Sharp ◽  
Valentine J Hyland ◽  
Ray Harris ◽  
...  
Keyword(s):  
Solid Phase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Pcr Primers ◽  
Clinical Samples ◽  
Hfe Gene ◽  
Target Sequence ◽  
Nucleotide Polymorphisms ◽  
Nitrophenyl Phosphate ◽  
Hemochromatosis Gene

Abstract Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples. Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. Results: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results. Conclusions: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.

scholarly journals First detection of Enterocytozoon bieneusi in whooper swans (Cygnus cygnus) in China

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Yuexin Wang ◽  
Kaihui Zhang ◽  
Yifan Zhang ◽  
Ke Wang ◽  
Azhar Gazizova ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Its Region ◽  
Enterocytozoon Bieneusi ◽  
Genetic Characteristics ◽  
Fecal Samples ◽  
Effective Strategies ◽  
Wide Range ◽  
Health Significance ◽  
Group 5 ◽  
Whooper Swan

Abstract Background Enterocytozoon bieneusi is a parasite that infects humans and a wide range of other animals. The large migratory waterfowl, the whooper swan (Cygnus cygnus), travels through many cities during its migration and can spread parasites. Despite receiving increasing attention worldwide, there have been no reports of E. bieneusi infection occurring in C. cygnus. Therefore, this study aims to assess the prevalence and genetic characteristics of E. bieneusi in C. cygnus in Sanmenxia, China. Methods Altogether, 467 fresh fecal samples were collected in the Swan Wetland Park in Sanmenxia, China. Genomic DNA was extracted from fresh fecal samples (n = 467) and E. bieneusi was identified by nested PCR amplification of the internal transcribed spacer (ITS) region. ITS-positive sequences were aligned and phylogenetically analyzed to determine the genotypes of E. bieneusi. Results The overall prevalence of E. bieneusi in C. cygnus was 7.49% (35/467). Sequencing of the 35 positive samples revealed eight known genotypes (EbpA, EbpC, Henan-III, Henan-IV, BEB6, CD9, Peru6 and PtEb IX) and three novel genotypes (CSW1, CSW2 and CSW3). The phylogenetic tree constructed from the ITS sequences showed that seven genotypes (Peru6, EbpA, EbpC, Henan-III, CSW3, Henan-IV and CSW1) clustered within the zoonotic Group 1 while the remaining novel genotype CSW2 clustered within Group 5. Conclusions To our knowledge, this is the first report of E. bieneusi in C. cygnus. Of public health significance, our results suggest that migratory C. cygnus might play an important role in the water-borne transmission of E. bieneusi. Effective strategies will be necessary to control E. bieneusi infection in C. cygnus, other animals and humans.

scholarly journals Evaluation of a Polymerase Chain Reaction-Based Heteroduplex Assay for Detecting the Adulteration of Processed Orange Juice with Mandarin Juice

2006 ◽  
Vol 89 (4) ◽  
pp. 1052-1060 ◽  
Author(s):  
Rachel Mooney ◽  
Louise Chappell ◽  
Angus I Knight
Keyword(s):  
Polymerase Chain Reaction ◽  
Orange Juice ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Target Sequence ◽  
Chain Reaction ◽  
Good Repeatability ◽  
Polymerase Chain ◽  
Mandarin Juice

Abstract A polymerase chain reaction (PCR)-based heteroduplex assay was evaluated for the detection of mandarin juice in processed orange juice. PCR amplification of a fragment of the chloroplast trnT-trnL intergenic spacer derived from mixtures of DNA extracted from orange and mandarin juice resulted in heteroduplex formation. The heteroduplex resulted from the co-amplification of a fragment containing an 8 base-pair indel that distinguished mixtures of orange and mandarin juice from orange juice and mandarin juice alone. The heteroduplex assay was evaluated against authentic juices obtained from different citrus species and confirmed that the marker was homogeneous within Citrus. The data obtained demonstrated maternal inheritance of chloroplast type in Citrus sp. and allowed the identification and confirmation of the maternal parentage of unknown and known citrus hybrids. Analysis of the quantitative potential of the PCR and polyacrylamide gel electrophoresis (PAGE) analysis demonstrated good repeatability with a coefficient of variation of 7.5%. Greatest sources of variance in experimental results were attributable to species and varietal differences in the levels of the PCR target. Mandarin juice contained approximately 18% (w/v) less PCR target sequence than did orange juice. The assay was tested in a blind trial using processed juices and correctly identified 20/22 samples with no false-positive results.

scholarly journals A PCR-based Technique for Identification of Fusicoccum sp. from Pistachio and Various Other Hosts in California

Plant Disease ◽  
2002 ◽  
Vol 86 (5) ◽  
pp. 515-520 ◽  
Author(s):  
Zhonghua Ma ◽  
Themis J. Michailides
Keyword(s):  
Host Plants ◽  
Extraction Procedure ◽  
Its Region ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Fungal Species ◽  
Pcr Assay ◽  
Shoot Blight ◽  
Dna Fragment ◽  
Polymerase Chain

Botryosphaeria panicle and shoot blight of pistachio, caused by Fusicoccum sp. is a destructive disease in California. In this study, a pair of group-specific polymerase chain reaction (PCR) primers BDI and BDII, was developed for identification of Fusicoccum sp. from pistachio and other hosts in California based on the sequences of the rDNA internal transcribed spacer (ITS) region. The primers amplified a 356-bp DNA fragment for all 73 tested isolates of Fusicoccum sp. collected from pistachio and other hosts throughout California in different years, but not for the other 33 fungal species isolated from pistachio and the eight isolates of Fusicoccum sp. obtained from pistachio trees in Greece. The PCR assay using this pair of primers was sensitive enough to detect 5 pg of genomic DNA of Fusicoccum sp. A simple DNA extraction procedure was developed that led to the rapid identification of Fusicoccum sp. from pistachio and other host plants in California.

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