Template For Polymerase Chain Reaction Research Articles

Design and application of universal primers for mitochondrial genome sequencing of synanthropic mite species (Astigmatina: Acaridae)</p >

This study aims to design and develop a set of universal primers for amplification of the entire mitochondrial genome of Acaridae mites based on polymerase chain reaction (PCR). The conserved regions were screened by comparing the mitochondrial genomes of known Acaridae mites in GenBank. To ensure that the primers cover the entire mitochondrial sequence, the mitochondrial genome was divided into six overlapping fragments based on the conserved region. After synthesizing primers, we extracted the total DNA of Acaridae mites collected from grain stores. Then the total DNA of individual Acaridae mites was used as the template for PCR, Sanger sequencing, and sequence splicing. Agarose gel electrophoresis revealed that the lengths of the six amplification products were between 1.2 kb and 3.8 kb, consistent with design expectations. The resulting sequences could be successfully spliced to form a loop structure, confirming that the primers cover the entire mitochondrial genome. The splicing results were consistent with the full sequence released in GenBank. Developed universal primers can achieve rapid amplification and help to obtain mitochondrial genome from a single acarid mite with high efficiency.

Development of a rapid quantitative method to differentiate MS1 vaccine strain from wild-type Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an economically important pathogen in the poultry industry. Vaccination is an effective method to prevent and control MS infections. Currently two live attenuated MS vaccines are commercially available, the temperature-sensitive MS-H vaccine strain and the NAD-independent MS1 vaccine strain. Differentiation of vaccine strains from wild-type (WT) strains is crucial for monitoring MS infection, especially after vaccination. In this study, we developed a Taqman duplex real-time polymerase chain reaction (PCR) method to identify MS1 vaccine strains from WT strains. The method was specific and did not cross-react with other avian pathogens. The sensitivity assay indicated that no inhibition occurred between probes or between mixed and pure templates in duplex real-time PCR. Compared with the melt-based mismatch amplification mutation assay (MAMA), our method was more sensitive and rapid. In conclusion, the Taqman duplex real-time PCR method is a useful method for the diagnosis and differentiation of WT-MS and MS1 vaccine strains in a single reaction.

Analisis filogenetik Hyposidra talaca nucleopolyhedrovirus (HytaNPV) yang diisolasi dari perkebunan teh Gunung Mas, Bogor, Jawa Barat dan virulensinya terhadap Hyposidra talaca Walker

Hyposidra talaca (Walker) is an important pest of tea plant. H. talaca can cause losses of between 40–100% in the dry season if proper control is not carried out. H. talaca has natural enemies such as predators, parasitoids, and pathogens. One of the entomopathogens is NPV. This study aims to obtain molecular characteristics through DNA polymerase sequences and determine the virulence level of NPV isolates from H. talaca. The same species from different locations can have genetic variability. Therefore, molecular characterization by polymerase chain reaction (PCR) on DNA polymerase sequences is one way to study the genetics of HytaNPV. NPV was isolated from infected H. talaca larvae collected from the field. The DNA isolates were used as templates for PCR for DNA polymerase gene amplification with an amplicon target of ±1,000 bp. A sequencing process followed the PCR provides nucleotide sequence. HytaNPV DNA polymerase sequencing results were aligned with GenBank’s BLAST data to provide information on the relationship of HytaNPV to NPVs isolated from other regions. Based on molecular character analysis using DNA polymerase gene sequence, HytaNPV Bogor has a homology level of 93.9% with HytaNPV isolated from India. HytaNPV Bogor has a genetic relationship with the NPV that infects Buzura suppressaria from China and Australia. HytaNPV Bogor is similar to the NPV that infects H. talaca from India. The bioassay of HytaNPV isolate against H. talaca showed the highest LT50 value of 1.92 days was found in concentration of 1.58 x 107 POBs/ml in second instar larvae.

Highly purified DNA-containing cell envelopes from fungi for direct use in PCR

Efficient DNA sample preparation from fungi with the rigid cell walls is still critical for successful polymerase chain reaction (PCR), one of the basic platforms in molecular diagnostics of fungi, especially in medical mycology. Common methods that involve different chaotropes to yield DNA samples have found a limited application for fungi. Here we describe a novel procedure for efficient production of permeable fungal cell envelopes with DNA inside as suitable templates for PCR. This procedure is facile, relies on boiling of fungal cells in aqueous solutions of selected chaotropic agents and additives and enables to remove RNA and proteins from PCR template samples. The use of chaotropic solutions containing 7 M urea, 1% sodium dodecyl sulfate (SDS), up to100 mM ammonia and/or 25 mM sodium citrate was the best option to yield highly purified DNA-containing cell envelopes from all fungal strains under study, including clinical Candida and Cryptococcusisolates. After treatment with the selected chaotropic mixtures, the fungal cell walls had undergone loosening and were no longer a barrier to release DNA in PCR as evident from electron microscopy examinations and successful target gene amplifications. Overall, the developed simple, fast, and low-cost approach to produce PCR-suitable templates in the form of DNA encased by permeable cell walls can find application in molecular diagnostics.

First report of maize rayado fino virus in corn fields in Santa Catarina, Brazil.

Maize (Zea mays L.) is the main cereal food of humans and animals in Brazil. In 2020 and 2021, a severe infestation of corn leafhoppers (Dalbulus maidis; Hemiptera: Cicadellidae) was observed in Santa Catarina State (South of Brazil). Subsequently, symptoms of chlorotic stripes limited in leaf veins started to appear in maize plants. Given the similarity of symptoms and the presence of high populations of corn leafhoppers in corn production areas, 30 plants in reproductive stage showing systemic symptoms were collected in summer and autumn from commercial fields of five municipalities in Santa Catarina: Campos Novos (27°23'18.0"S, 51°12'52.7"W), Lages (27°47'17.8"S, 50°18'16.9"W), Mafra (26°06'42"S, 49°48'25"W), Fraiburgo (27°01'36"S, 50°55'19"W), and Abelardo Luz (26°34'02"S, 52°20'02"W). The young leaves of these samples were used for molecular analyses targeting the maize rayado fino virus (MRFV; Tymoviridae: Marafivirus). Total nucleic acids were extracted using TRIzol® (Invitrogen, USA), following the manufacturer's instructions. These were used as a template for cDNA synthesis with the enzyme MMLV-RT (Promega, USA), following the manufacturer's instructions. The polymerase chain reaction (PCR) was performed using Gotaq® DNA polymerase (Promega, USA) and MRFV-09/MRFV-10 primers (Hammond et al. 1997). All PCR products were subjected to electrophoresis in 1% agarose gel and were visualized under ultraviolet light. Twenty-eight of the 30 tested plants were MRFV-positive, showing a fragment with an expected size of ~633 bp. To confirm our results, all MRFV-positive samples were sent for sequencing (GenBank accession numbers OM763708 - OM763710 and ON730784 - ON730806) and submitted to BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi), resulting in identities ranging from 96.21% to 99.21% with the isolate "Brazil 26" of MRFV, which was detected in 2005 in São Paulo, Brazil (GenBank accession nº: AF186178) (Hammond and Bedendo 2005). A second set of primers was used to validate the first PCR, confirming MRFV infection (data not shown).Moreover, whitish streaks and leaf reddening were observed on the leaves of some plants; therefore, the identification for phytoplasmas (Candidatus Phytoplasma asteris) and spiroplasmas (Spiroplasma kunkelii) from the corn stunt complex was performed. For this, previously extracted nucleic acids from each sample were used as templates for a multiplex PCR using the primers CSSR6/CSSF2 and R16F2n/R16R2 (Gundersen and Lee 1996; Barros et al. 2001). Two plants were infected with only spiroplasma, 17 samples were infected with Spiroplasma and MRFV, and three samples were infected by these three pathogens. An increasing incidence of corn stunt has been observed in commercial fields in Santa Catarina in recent years. Mollicutes are commonly found and mostly studied as causal agents of corn stunt disease. On the contrary, despite being present in Brazil since the 1970s, the virus is less studied because its contribution to the corn stunt complex is still unknown (Hammond and Bedendo 2001). In this report, indications that the virus is expanding to different regions in southern Brazil were observed, which raises an opportunity for further evaluation and its consideration in monitoring programs. Moreover, to the best of our knowledge, this is the first report of MRFV in Santa Catarina, Brazil.

Molecular characterization of Streptococcus agalactiae and Streptococcus dysgalactiae causing bovine mastitis in the southern region of Bangladesh.

This study was conducted to validate polymerase chain reaction (PCR) as a confirmatory diagnostic tool to find out the presence and frequency of Streptococcus agalactiae (S. agalactiae) and Streptococcus dysgalactiae (S. dysgalactiae) in mastitic milk samples obtained from dairy cows in the southern region of Bangladesh. A total of 196 samples of bovine milk were collected from various dairy farms in the Chattogram metropolitan area of the southern part of Bangladesh. DNA extracted from isolates obtained by culturing California mastitis test (CMT)-positive mastitic milk samples (n = 146) on 5% sheep blood agar was used as a template for PCR. Two sets of specific primers based on the 16S rRNA gene were used to discriminate between S. agalactiae and S. dysgalactiae. Four PCR products were subjected to sequencing, followed by phylogenetic analysis. The PCR analyses revealed that out of the 146 CMT-positive milk samples tested, 29 samples were positive for S. agalactiae (19.86%), while 26 samples were positive for S. dysgalactiae (17.81%). Further sequence analysis of the corresponding PCR products and bioinformatics analysis verified the results. The study proves the efficiency of PCR as a useful diagnostic approach to determine the presence and prevalence of S. agalactiae and S. dysgalactiae in mastitic milk samples obtained from dairy cows.

Identification of the PLAG1 polymorphisms in Peranakan Ongole cattle population

Genetic factor plays a very important role in determining of cattle performance. A gene which is reported to be controlling cattle growth and body size is pleomorphic adenoma gene 1 (PLAG1). Therefore, the goal of this study was to identify PLAG1 polymorphisms in PO cattle population. A total of 43 unproductive PO cows from the slaughterhouse at Kebumen were used in this study. Blood samples were collected individually when the cattle were slaughtered. Furthermore, DNA genome was harvested from blood samples and used as DNA template for polymerase chain reaction (PCR) targeting g.48308 C>T, g.32212 (19 bp indel) and g.45233 T>C polymorphisms. g.48308 C>T and g.45233 T>C SNPs were genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) technique and a 19 bp indel of PLAG1 was directly genotyped by observing PCR products. Both allele and genotype frequencies were calculated to test Hardy-Weinberg equilibrium (HWE) status. The results showed that g.48308 C>T SNP and 19 bp indel varied. Three genotypes of the g.48308 C>T SNP namely CC, CT and TT were found in this study and two genotypes (DD and DI) of 19 bp indel were also identified. Moreover, heterozygous CT genotype frequency of g.48308 C>T SNP was 0.51 and T allele frequency was 0.60. In term of 19 bp indel, DD genotype frequency was 0.88 and D allele frequency was 0.94. In conclusion, two polymorphisms of the PLAG1 were identified in PO cattle population.

Fast and more affordable CYP2C19 LOF testing to personalized of clopidogrel therapy

Abstract Background Clopidogrel is an antiplatelet drug used to treat coronary artery disease (CAD) to reduce blood clotting by inhibiting platelet activity. However, many patients experience ischemic events due to changes in clopidogrel metabolism attributed to various genetic and non-genetic factors. CYP2C19 is one of the important genes responsible for the metabolism of clopidogrel, but this gene has polymorphisms in cytochrome P450 (CYP), and this CYP2C19 Loss-of-function (LoF) polymorphisms impair clopidogrel metabolism between individuals and races. Therefore, genetic testing is an effective strategy to personalized clopidogrel and reduce the incidence of ischemic events in CAD patients. Purpose We sought to develop a nested allele-specific multiplex polymerase chain reaction (PCR) method for the detection of CYP2C9 LOF alleles. Method Genomic DNA was extracted from blood taken from 7 healthy volunteers using a commercial DNA extraction kit. A two-step PCR method was developed. First, DNA was subjected to a first PCR used to amplify exons 4 and 5 simultaneously in a single reaction tube. Second, The products of 1st PCR were then used as a template in the second PCR to detect CYP2C9 polymorphisms alleles (CYP2C19*2 and *3) using allele-specific primers. The test results were validated via sequencing. Result We successfully detected clinically important CYP2C19 LOF polymorphisms alleles by the nested allele-specific multiplex PCR method (Figure 1). This method is fast and doesn't require high-quality and expensive laboratory equipment. It was reproducible and specific and can reliably detect CYP2C19 variants. Direct sequencing of target variants has been used to validate this strategy further, and the amplified sequences were 100% identical to the CYP2C9 sequence. Conclusion The developed methods were specific to identify polymorphisms in CYP2C19 (rs4244285 and rs4986893). However, to understand ethnic differences and their potential implications for clopidogrel therapy, we highly recommend identifying relevant genetic polymorphisms before clinical manifestations arise. Funding Acknowledgement Type of funding sources: None.

P455 Identification of Cryptic species of Aspergillus using Beta-tubulin gene in a tertiary care center in South India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesTo identify cryptic species of the genus Aspergillus using Beta-tubulin gene by sequence typing.Methods Aspergillus grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud's Dextrose Agar/Oatmeal Agar.Tease mount/Slide culture was done to study the morphological features of the hyphae, size, shape, and arrangement of the conidia.DNA Extraction using phenol-chloroform method: DNA was extracted and purified directly from 4-day-old culture and used as a template for polymerase chain reaction (PCR) amplification.DNA Amplification: A fragment of the target gene were amplified using PCR.The primer pairs used for Beta-tubulin gene was Bt2a (Forward primer) and Bt2b (Reverse primer).Beta-tubulin.Bt2a FGGTAACCAAATCGGTGCTGCTTTCBt2b RACCCTCAGTGTAGTGACCCTTGGCDNA Purification: The PCR product was purified with multi-screen filter plates.Gel electrophoresis: The amplified DNA product was subjected to agarose gel electrophoresis and specific band formation was to be observed for the species of Aspergillus.DNA Sequencing: The purified product will be used as a template for sequencing. An applied biosystem 3730 sequencer will be used to obtain DNA sequences.Phylogenetic Analysis: Parsimony analysis of individual and combined matrices will be conducted using PAUP version 4.0b10 software/free software at phylogeny.fr by the maximum parsimony (MP) and Neighbor-joining methods.The results will be represented using a dendrogram.ResultsA total of 30 Aspergillus isolates were collected and identified using Phenotypic methods.The DNA extraction and PCR amplification were done and sent for Sequencing. The results of Sequence typing are awaited.ConclusionCryptic species are morphologically indistinguishable forms of Aspergillus and their identifications can be confirmed exclusively by using molecular techniques which have led to the description of previously unknown or rare species among different Aspergillus species complex. According to studies, the frequency of such species was found to be 10% in TRANSNET study U.S and 12% in FILPOP study Spain. Most of these species are less susceptible and certain species are multidrug-resistant (azoles and amphotericin B) and the emergence of antifungal-resistance among the Aspergillus species forms the importance in identifying Cryptic species.

Detection of Cytomegalovirus in Urine Specimen of Cholestatic Infants by Polymerase Chain Reaction

Cholestatic infants are associated with congenital abnormalities or viral infections, such as cytomegalovirus (CMV) infection. CMV can be detected by polymerase chain reaction (PCR) in body fluids, including urine which can be obtained easily and is non-invasive. The objective was to detect CMV in urine specimens of cholestasis infants and to analyze its correlation with serological status. This was a descriptive observational study with the cross-sectional approach, used urine from 39 cholestatic infants who meet the inclusion and exclusion criteria and have been approved by Ethics Committee. The nested-PCR was performed from extracted urine and unextracted direct urine. Serological data of immunoglobulin (Ig) M and IgG data were collected. Data were analyzed by Chi-square. Detection of CMV from extracted urine by PCR showed positive in 87.2% patients and from unextracted urine was positive in 48.7% patients. Serological status showed that IgM was positive in 41.0% patients and IgG was positive in 89.7% patients. The acute infection (IgM+ IgG+) was found in 41.0% patients, past infection (IgM-IgG+) was 48.7% patients, and not infected (IgM-IgG-) was in 10.3% patients. The acute infection (IgM+ IgG+), past infection (IgM-IgG+) and not infected (IgM-IgG-) was found in 41.0%, 48.7%, and 10.3% patients, respectively. The correlation between PCR CMV from extracted urine with serological CMV was moderate, while the unextracted urine was low. It indicates that to detect the infection of CMV, PCR technique is more accurate than serological testing, and the extracted urine is more appropriate specimen as PCR template than direct urine.

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

DESCRIPTION OF THE FIRST KLOSSIA SPECIES (APICOMPLEXA: EUCOCCIDIORIDA: ADELEORINA: ADELEIDAE) INFECTING A PULMONATE LAND SNAIL, TRIODOPSIS HOPETONENSIS (MOLLUSCA: POLYGYRIDAE), IN NORTH AMERICA.

Snails identified as Triodopsis hopetonensis were collected (n = 18) from the University of Arkansas-Fayetteville campus in December 2018. Additional snails were collected in April 2019 (n = 9) and in September 2019 (n = 9). Kidney tissues were examined using light microscopy, and polysporocystic oocysts were observed. Sporulated oocysts (n = 2) measured 78 ± 3.4 μm × 76 ± 2.9 μm with an irregular oocyst residuum and contained an estimated 44-55 tetrazoic sporocysts. The sporocysts (n = 10) measured 13 ± 0.5 μm × 11 ± 1.5 μm with an indistinct, irregularly placed, sporocyst residuum and usually contained 4 sporozoites, although an octozoic variant was observed. DNA was extracted from the snail kidney tissues and used as a template for polymerase chain reaction (PCR). PCR was used to determine the infection status of the snails; 13 of 36 (36%) specimens were found to be infected with a new Klossia species, and only 3 (8%) of these infections were detected using light microscopy. The complete nuclear 18S ribosomal DNA (1,800 bp) and mitochondrial genomes (6,775 bp) were generated, and they differentiated this parasite from the type species Klossia helicina and support the description of this new Klossia species, Klossia razorbacki n. sp. This is the first Klossia species to be described from any North American snail.

Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection.

Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Small-Scale Preparations of Yeast DNA.

In this protocol, yeast DNA is prepared by digestion of the cell wall and lysis of the resulting spheroplasts with SDS. This method reproducibly yields several micrograms of yeast DNA that can be efficiently cleaved by restriction enzymes and used as a template in polymerase chain reaction (PCR). Note that yeast colonies can also be used directly in PCR, without purifying yeast DNA.

Genetic identification of Chinese drug materials in yams (Dioscorea spp.) by RAPD analysis

The botanical origins of Chinese drug materials in yams were examined among eighteen cultivarities/ species of Dioscorea by randomly amplified polymorphic DNA (RAPD). DNA isolated from fresh leaves were used as a template in polymerase chain reactions (PCR) using thirty random decamer primers. Eighteen primers produced 237 polymorphic RAPD markers, which were scored for all the lines studied. Four primers including OPP-04, OPP-15, OPAP-04 and OPAP-10, could distinguish all tested samples. Genetic distances and similarities among eighteen cultivarities/species were calculated, and a cluster analysis was used to generate a dendrogram showing a phylogenetic relationship. The studied Dioscorea species could be divided into five subgroups by phylogenetic analysis. Genetic distance and similarity analysis showed that line 70W37 was distantly related to 70W47, and line 70W41 was closely related to 70W59. RAPD technique could be successfully applied for the rapid identification and differentiation of Dioscorea species.

One-Step Bacterial Artificial Chromosome (BAC) Modification: Cloning of the A Homology Arm into Reporter-Shuttle Vector.

In this protocol, the homology arm sequence for one-step bacterial artificial chromosome (BAC) modification is introduced by ligation into the shuttle vector carrying the reporter sequence to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector. To provide further assurance that the homology box has been successfully integrated into the plasmid, the enzyme digestion pattern of the modified plasmid is compared with that of the unmodified plasmid.

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Cloning of the A and B Homology Arms into the Shuttle Vector.

This protocol describes the preparation of the shuttle vector before its introduction into bacterial artificial chromosome (BAC) host cells for BAC two-step engineering. The homology arm sequences, prepared previously, are introduced by ligation into the digested shuttle vector DNA to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector.

Sensitivity of serological and polymerase chain reaction methods for detection of viruses in Allium spp.

Infection of Onion yellow dwarf virus (OYDV), Shallot yellow stripe virus (SYSV), and Garlic common latent virus (GCLV) has been reported on Allium spp. in Indonesia. Serology methods using enzyme-linked immunosorbent assay (ELISA), and dot immunobinding assay (DIBA) is a common method to detect viruses, while polymerase chain reaction (PCR) is popular as a molecular method to detect plant viruses nowadays. This research was conducted to assess the sensitivity of 3 detection methods, i.e. ELISA, DIBA and PCR for detecting viruses in Allium spp. Sensitivity level of each method was evaluated by diluting plant extract and antibody for ELISA and DIBA, and cDNA template for PCR. Result of this research showed that DIBA was able to detect OYDV, GCLV, and SYSV with plant extract dilution in the range of 10−1 to 10−2, and antibody dilution from 1:300 to 1:1000. ELISA seems to be more sensitive than DIBA because it was able to detect the same virus with plant extract dilution in the range of 10−1, 10−3, and 10−5, and antibody dilution from 1:300 to 1:1000. RT-PCR method was the most sensitive method compared to ELISA and DIBA because it has the highest accuracy, specification and sensitivity level. Specific DNA fragment was amplified using universal primers for Potyvirus and Carlavirus up to 10−3 and 10−4 dilution factor of cDNA respectively. Moreover, amplification using specific primers of OYDV, SYSV, and GCLV is more sensitive than using universal primers, because up to 10−5 dilution factor of cDNA was able to amplify virus target.

First Report of Strawberry (Fragaria × ananassa) as a Host of a ‘Candidatus Phytoplasma americanum’-Related Strain in the United States

In June 2018, while conducting a survey for possible presence of phytoplasma pathogens in Pennsylvania production fields, a strawberry plant (Fragaria × ananassa) was observed with symptoms of stunting and unseasonal reddening and distortion of leaves. Only one out of 500 strawberry plants expressed the described symptoms in a farm in Perry County, PA. Several leaves and stems were collected from the symptomatic plant as well as from a few asymptomatic ones for further work. Total DNA was extracted from 100 mg of leaf midveins with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). All DNA samples were initially tested using a phytoplasma universal real-time polymerase chain reaction (PCR) assay (Hodgetts et al. 2009). Positive amplification results were obtained only with samples derived from the symptomatic strawberry plant. To detect and identify the suspected phytoplasmal pathogen strain(s), the DNA samples were used as a template in conventional PCRs for amplification of phytoplasma 16S rRNA, secY, secA, and tuf gene sequences as previously described (Dickinson and Hodgetts 2013; Lee et al. 2004, 2010; Makarova et al. 2012). All of the obtained amplicons were of expected sizes, and no amplification was observed from the asymptomatic strawberry samples. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen) and sequenced. Nucleotide sequences of the PCR products were deposited in the GenBank database under accession numbers MN227133 to MN227136. The nucleotide sequences were analyzed using Geneious software (Biomatters, Auckland, NZ) for sequence alignments and using iPhyClassifier (Zhao et al. 2009) for group-subgroup and ‘Candidatus Phytoplasma’ species assignments. The classification into 16Sr groups and subgroups was established by virtual restriction fragment length polymorphism (RFLP) analysis of PCR products compared with reference strains. The phytoplasma, termed strain SRL1-PA (Strawberry Red Leaf1-PA), was found to share 99.9% similarity with that of the ‘Candidatus Phytoplasma americanum’ reference strain (GenBank accession DQ174122). The virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is most similar to the reference group XVIII, subgroup A (GenBank accession DQ174122), with a pattern similarity coefficient of 0.98. Another sample from the same strawberry plant was independently determined positive for ‘Ca. P. americanum’ by CPHST Beltsville Lab using nucleotide sequence analysis of 16S rRNA gene sequences amplified in end point PCR. Alignments and phylogenetic analyses of secY, secA, and tuf gene sequences confirmed the close relatedness of the Pennsylvania SRL1-PA strain with the reference strains of ‘Ca. P. americanum’. ‘Ca. P. americanum’ was previously reported (Lee et al. 2006) in Texas and Nebraska on potato and associated with potato purple top wilt disease. In 2019, the Pennsylvania Department of Agriculture team revisited the same farm and collected additional samples including some from potato but was not able to detect any phytoplasma. To our knowledge, this report provides the first evidence for the presence of a ‘Ca. P. americanum’-related strain in Pennsylvania and more importantly describes strawberry (Fragaria × ananassa) as a new host for this phytoplasma. With only one single positive strawberry plant in a single year, the true extent of disease spread on strawberries remains undetermined.