Polymerase Chain Reaction Primer Sets Research Articles

NEW PRIMER DESIGN of MULTIPLEX POLYMERASE CHAIN REACTION (PCR) for DETECTION of Escherichia Coli and Salmonella Enterica in FOOD SAMPLE

Background: Multiplex PCR techniques are used to detect multiple pathogens at the same time simultaneously. In order to achieve the specificity and sensitivity of detection, it is important to optimize the multiplex PCR method properly. Developing suitable primers and optimizing PCR temperature to boost particular genes from pathogens are key factors for this optimization. Methods: For the simultaneous detection of Escherichia coli and Salmonella enterica in food samples, this research aims to develop a set of primers for multiplex polymerase chain reaction (PCR). Using Primer-BLAST software, the study utilizes specific primer designs for the phoA gene (Eschericia coli) and invA gene (Salmonella enterica). DNA isolation has confirmed successful extraction from the two bacterial samples. PCR was performed under different conditions, including Singleplex and Multiplex PCR, using two annealing temperatures of 53oC and 50oC. Results: The results showed that this method can effectively amplify target genes and indicate their specificity and reliability at both temperature levels. Given these results, it has successfully conducted multiplex PCR using the built primer pairs. Both annealing temperatures of 50oC and 53oC can be used to perform multiplex PCR to detect E. coli and Salmonella enterica in one PCR reaction. Conclusion: Through this research, we have created a new set of Multiplex PCR Primers and an optimized multiplex PCR technique for the simultaneous detection of E coli and Salmonella enterica in food samples. The rapid and simultaneous screening of E. coli and S. enterica, which contributes to improved food safety measures and pathogen detection in the food industry, is promising with this optimized PCR approach.

A novel qPCR-based technique for identifying avian sex: An illustration within embryonic craniofacial bone.

Sex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female-specific W chromosome has previously been identified in chick using a species-specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT-qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide-binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT-qPCR primer set was confirmed in all three species using species-specific PCR, including a novel quail-specific HINTW PCR primer set. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT-qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.

Real Time PCR-based diagnosis of human visceral leishmaniasis using urine samples.

Diagnosis of visceral leishmaniasis (VL) through the detection of its causative agents namely Leishmania donovani and L. infantum is traditionally based on immunochromatographic tests, microscopy of bone marrow, spleen aspirates, liver or lymph node and differential diagnosis. While the first process has low specificity, the later one carries the risk of fatal hemorrhage. Over the last decade, multiple Polymerase Chain Reaction (PCR) based diagnosis has been developed using blood and urine sample with a varying degree of sensitivity and specificity, an issue worth improving for precision diagnosis. Earlier, we reported a PCR-based diagnosis of L. donovani in peripheral blood using a novel set of PCR primers with absolute specificity. Using the same set of primers and PCR conditions, here we describe diagnosis of L. donovani from urine, for a non-invasive, rapid and safe diagnosis. Diagnosis of VL was carried out using urine samples collected from clinically diagnosed VL patients (n = 23) of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients (n = 19). The method showed 100% sensitivity in detecting L. donovani in urine and corresponding blood and retrospective bone marrow samples, as well as 100% specificity in control groups. A Real Time PCR-based molecular detection system using urine sample is hereafter presented what could be a, non-invasive approach for VL detection with precision and perfection.

Comparison and Selection of Conventional PCR Primer Sets for Studies Associated with Nitrogen Cycle Microorganisms in Surface Soil

The nitrogen cycle is a biogeochemical cycle primarily associated with the microbial activity that occurs in various environments, including soil. Various genes related to the nitrogen cycle have been studied for different purposes by many researchers; therefore, the polymerase chain reaction (PCR) conditions and gene compositions differ among reports, making comparisons difficult. In this study, we compare the PCR methods to amplify 13 nitrogen cycle-related genes (amo (amoA and amoB), norB (cnorB and qnorB), hzs, napA, narG, nifH, nirK, nirS, nosZ, nrfA, and nxrA) in the soil samples collected from four land use types and selected a method with excellent applicability. However, the PCR method for five nitrogen cycle-related genes (amoC, hao, hzo, nirB, and nxrB) could not be presented. In addition, the nitrogen cycle-related genes from the four land use types (field, forest, bare land, and grassland) and the seasonally collected samples were analyzed and discussed. In the grassland samples, all the nitrogen cycle-related genes reviewed were amplified. These results vary from those of the field, forest, and bare land samples, and it was estimated that grassland, among the land use types, could play an important role in the nitrogen cycle in soil. However, an association between the seasons and the rainy season was not confirmed. Thus, this study may be used for future research in various fields related to the nitrogen cycle.

Rapid Diagnostic Testing for Response to the Monkeypox Outbreak - Laboratory Response Network, United States, May 17-June 30, 2022.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

Complementary combination of multiplex high‐throughput DNA sequencing for molecular phylogeny

AbstractThe rapid development of DNA sequencing technology in recent years has provided new tools for phylogenetic data acquisition. By using high‐throughput DNA sequencing technology, molecular phylogenetic information can be obtained more quickly and economically. Here, we describe a complementary combination of two multiplex high‐throughput DNA sequencing methods. One is multiplexed phylogenetic marker sequencing (MPM‐seq), and the other is multiplexed inter‐simple sequence repeat (ISSR) genotyping by sequencing (MIG‐seq), whose protocol is improved over that of the original one. Both MPM‐seq and MIG‐seq begin with multiplex polymerase chain reaction (PCR), each amplifying multiple phylogenetic markers and genome‐wide ISSR regions, respectively. After another PCR using a second PCR primer set that is common in both methods, next‐generation sequencing is used to simultaneously detect DNA sequences of multiple regions from multiple samples in each method. In this case study, we performed a molecular phylogenetic analysis of Japanese fir (Abies) and the closely related Abies species. MPM‐seq revealed DNA sequences of three regions from chloroplast DNA and one nuclear internal transcribed spacer and created a partially informative phylogenetic tree for 13 Abies species. Whereas MIG‐seq detected 6700 single‐nucleotide polymorphisms and exhibited clear clustering of related species with 97%–100% bootstrap support for all branches of the phylogenetic tree. Hence, with a complementary combination, quick, simple, and economical analysis can be performed in a wide range of genomic studies, including molecular phylogeny, as well as for investigating genetic differentiation or genetic identification among species, hybrids, and populations, and even among clones and cultivars, as a DNA barcoding technique.

Emerging Pathogenic Gammaproteobacteria Wohlfahrtiimonas chitiniclastica and Ignatzschineria Species in a Turkey Vulture (Cathartes aura).

New World vultures, such as turkey vultures (Cathartes aura), are obligate scavengers with large geographic ranges. In a preliminary characterization of the turkey vulture (TV) gastrointestinal microbiome in Southern California, we identified 2 recently described emerging bacterial pathogens not previously known to be associated with this avian species. High-throughput sequencing of broad-range 16S rRNA gene amplicons revealed sequences from TV cloacal swabs that were related closest to Wohlfahrtiimonas chitiniclastica and Ignatzschineria species, both Gammaproteobacteria considered by the United States Centers for Disease Control and Prevention as emerging zoonotic pathogens. None of these bacterial sequence types have been previously identified from samples obtained from the turkey vulture gastrointestinal microbiome. With the use of bioinformatics workflows previously established by our research group, we designed specific and sensitive polymerase chain reaction primer sets that represent novel diagnostic assays for the genera Wohlfahrtiimonas and Ignatzschineria. These primer sets were validated by Sanger sequence confirmation from complex TV samples. Because the genera Wohlfahrtiimonas and Ignatzschineria are both known to have dipteran hosts, the molecular diagnostic tools we present here should be useful for better understanding the role of flies, vultures, and other scavengers in the ecology and epidemiology of the genera Wohlfahrtiimonas and Ignatzschineria from a One Health perspective.

Association between ALFPm3 single nucleotide polymorphism and white spot syndrome virus resistance in black tiger shrimp Penaeus monodon.

Here single nucleotide polymorphisms (SNPs) were associated with white spot syndrome virus (WSSV) resistance in black tiger shrimp Penaeus monodon. SNPs were identified by single-strand conformation polymorphism (SSCP) screening and DNA sequencing of shrimp sampled from 3 families (100 shrimp per family) challenged with WSSV. Shrimp that died over the 14 d challenge trial were designated susceptible, with those remaining alive on Day 14 designated resistant. To compare SNPs, 10 samples from the susceptible and resistant groups, each comprising DNA pooled from 3 shrimp, were amplified by polymerase chain reaction (PCR) using primers to 12 selected genes and screened by SSCP. SNPs were only identified in the anti-lipopolysaccharide factor 3 (ALFPm3) gene product. Analysis of complete ALFPm3 gene sequences confirmed the existence of 3 SNPs (g.934C>G, g.1186A>G, and g.1898C>G) that were polymorphic between the susceptible and resistant groups. Further analyses using specific tetra-primer amplification refractory mutation system PCR primer sets associated these 3 SNPS, and particularly the g.1186A>G SNP, with WSSV resistance. This SNP thus has potential for use as a DNA marker to select for WSSV resistance in P. monodon breeding programs.

Polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses.

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

Comparison of PCR Primers for Analyzing Denitrifying Microorganisms in the Hyporheic Zone

In this study, the specific amplifications of six denitrification-associated genes using PCR(Polymerase Chain Reaction) primer sets were compared. Thereafter, the PCR primer sets that were determined to be suitable for each denitrification-associated gene were used to test samples from sixteen aqueous environments (three from groundwater, three from stream water, and ten from hyporheic zone water). The specific amplification was determined using PCR primer sets for denitrification-associated genes and nucleic acids from eleven types of strains. NosZ was the most frequently amplified gene from the nucleic acid of type, with a specific band seen in all eleven strains. The specific band amplification and PCR time of the strains were analyzed to select one PCR primer set for each gene. The selected PCR primer sets were used to analyze sixteen samples from the aqueous environments in which denitrifying microorganisms were expected to be present. Specific bands of narG, nirS, and nosZ were most frequently observed in the hyporheic water samples. The results showed that microorganisms containing nirG (involved in the reduction of nitrate to nitrite), nirS (involved in the reduction of nitrite to nitric oxide), and nosZ (involved in the reduction of nitrous oxide to nitrogen gas) were the most abundant in the hyporheic zone samples used in this study.

Reclassification of a frequent African-origin variant from PMS2 to the pseudogene PMS2CL.

Genomic analysis has become a mainstay in the investigation of cancer patients, especially for those suspected of harboring a heritable cancer predisposition syndrome. With ubiquitous short-read next-generation sequencing (NGS) technologies, these analyses can be complicated by the inappropriate alignment of variants to homologous genomic regions or pseudogenes. Using distinct primer sets specific to the gene and pseudogene, a nonspecific primer set, and a highly gene-specific long-range polymerase chain reaction primer set, we have shown that in at least a subset of patients, the common African PMS2 variant NM_000535.5:c.2182_2184delACTinsG, classified as pathogenic in ClinVar, has been incorrectly assigned to PMS2 from its well-documented pseudogene, PMS2CL. This result is not only important for patients but also highlights a weakness in short-read NGS technologies and the racial inequity in genomic analysis.

Pasteurellaceae members with similar morphological patterns associated with respiratory manifestations in ducks.

Aim:A total of 112 freshly dead ducks aged from 2 to 20 weeks old with a history of respiratory manifestations were investigated for the implication of Pasteurellaceae family members.Materials and Methods:Isolation and identification to the family level were conducted by conventional bacteriological methods, including microscopic examination and biochemical characterization. Identification to the species level was conducted by polymerase chain reaction (PCR) and analytical profile index (API) 20E kits.Results:Conventional bacteriological isolation and biochemical characterization revealed the infection of 16/112 examined birds with a prevalence rate of 14.3%. PCR confirmed the detection of Pasteurellaceae family conserved genes RpoB and Bootz in 16/16 (100%) isolates. PCR was also used for genus and species identification of the isolated Pasteurellaceae members; the results revealed that 5/16 (31.3%) of isolates were Gallibacterium anatis and 2/16 of isolates (12.5%) were Pasteurella multocida. Riemerella anatipestifer, Mannheimia haemolytica, and Avibacterium paragallinarum were not detected by PCR. Biotyping by API 20E successfully identified 5/16 (31.3%) isolates that could not be typed by PCR and confirmed their belonging to Pasteurella pneumotropica. Neither the available PCR primer sets nor API 20E succeeded for species identification of 4/16 (25%) isolates. Antibiotic susceptibility profiling of isolates revealed that 16/16 (100%) of isolates demonstrated multidrug resistance (MDR) phenotypes. Moreover, 16/16 (100%) of isolates demonstrated a phenotypic resistance pattern to neomycin.Conclusion:Combined genotypic, phenotypic, biotyping, and virulence characterizations are required for laboratory identification of pathogenic Pasteurellaceae. Moreover, P. multocida was not the prevailed member implicated in respiratory problems in ducks as P. pneumotropica, G. anatis, and unidentified strains were involved with higher prevalence. Chloramphenicol and ampicillin demonstrated the highest in vitro effects on the studied Pasteurellaceae. Furthermore, the prevalence of multidrug-resistant isolates signified the demand to implement targeted surveillance in the ducks’ production sector, and MDR survey in poultry sectors in Egypt to apply effective control measures.

PCR Assays Based on invA Gene Amplification are not Reliable for Salmonella Detection

Background: Salmonella surveillance relies on invA polymerase chain reaction (PCR) assays for the rapid detection of Salmonella; however, false-positive results have been reported using this method. Objectives: To evaluate the performance and specificity of the published and validated PCR protocols targeting invA gene for the detection of Salmonella. Methods: The performance and specificity of 11 different PCR primer sets were evaluated using Salmonella type strains and Citrobacter spp., Escherichia coli and Serratia spp. isolates recovered during a Salmonella surveillance program. Results: It was revealed that the published PCR protocols using validated primers targeting invA and 16S rRNA genes generated false-positive signals. Importantly, a protocol targeting the ttrA/C genes was able to discriminate Salmonella and non-Salmonella isolates. Conclusions: Detection of Salmonella spp. by means of invA PCR amplification is not reliable. In fact, false-positive results are commonly obtained from Citrobacter, E. coli and Serratia isolates. It is recommended to use other loci, such as ttrA/C genes, for the accurate and reliable detection of Salmonella.

Quantitative assessment of Lactococcus lactis subsp. cremoris present in artisanal raw cow’s milk cheese

Lactococcus lactis subsp. cremoris belongs to lactic acid bacteria that play a crucial role in cheese production and it is known to be beneficial to human health. The aim of the study was to establish a rapid and accurate quantitative real-time polymerase chain reaction (qPCR) method to detect and enumerate L. lactis subsp. cremoris in artisanal raw cow’s milk cheese. Artisanal raw cow’s milk cheese samples were used to check for presence and number of L. lactis subsp. cremoris strains. The method applies a set of target-specific PCR (polymerase chain reaction) primers and a fluorogenic probe, and amplifies a part of the LACR_RS01280 gene that encodes the aminoacetone oxidase family flavin adenine dinucleotide (FAD) binding enzyme. All 5 L. lactis subsp. cremoris strains examined were found to be qPCR positive. There was no signal recorded for 8 strains which belong to closely related species. The limit of detection amounted to ten copies per reaction and the assay indicated a linear dynamic range of seven logs. This method may be applied in detection and enumeration of L. lactis subsp. cremoris in cheese during its ripening. Moreover, it may be applied to examine the distribution of L. lactis subsp. cremoris during the cheese production and ripening.

Gelatin speciation using real-time PCR and analysis of mass spectrometry-based proteomics datasets

Authentication of gelatin, an ingredient in food and drug products, is of interest with respect to some regulations and religion rules. Determination of the gelatin origin is particularly challenging because of the similarity in amino acid sequences of collagen types in different species. In this study, Polymerase Chain Reaction (PCR) technique using species-specific primers and also chemometrics analysis of mass spectral data were investigated to differentiate bovine, porcine and fish source of gelatin in commercial pure gelatin and gelatin-containing food and drug products. The specific PCR primer set was optimized using real-time PCR approach to routinely determine the gelatin source. Real-time PCR is sensitive to identify gelatin origin from traces of species-specific DNA. However, source determination is impossible when DNA is denatured or removed during production process. Alongside, chemometrics methods (PLS/DA and PCA) were used for mining LC/MS data sets to obtain patterns for discrimination of gelatin origins. LC/MS data sets involve MS spectrum of tryptic gelatin peptides generated from gelatin samples. Using PLS/DA method, a discrimination model is developed for identifying origin of the extracted gelatin. The method is practical to determine the halal authenticity of gelatin in the pure and processed products.

The Detection and Characterization of QoI-Resistant Didymella rabiei Causing Ascochyta Blight of Chickpea in Montana.

Ascochyta blight (AB) of pulse crops (chickpea, field pea, and lentils) causes yield loss in Montana, where 1.2 million acres was planted to pulses in 2016. Pyraclostrobin and azoxystrobin, quinone outside inhibitor (QoI) fungicides, have been the choice of farmers for the management of AB in pulses. However, a G143A mutation in the cytochrome b gene has been reported to confer resistance to QoI fungicides. A total of 990 isolates of AB-causing fungi were isolated and screened for QoI resistance. Out of these, 10% were isolated from chickpea, 81% were isolated from field peas, and 9% isolated from lentil. These were from a survey of grower’s fields and seed lots (chickpea = 17, field pea = 131, and lentil = 21) from 23 counties in Montana sent to the Regional Pulse Crop Diagnostic Laboratory, Bozeman, MT, United States for testing. Fungicide-resistant Didymella rabiei isolates were found in one chickpea seed lot each sent from Daniels, McCone and Valley Counties, MT, from seed produced in 2015 and 2016. Multiple alignment analysis of amino acid sequences showed a missense mutation that replaced the codon for amino acid 143 from GGT to GCT, introducing an amino acid change from glycine to alanine (G143A), which is reported to be associated with QoI resistance. Under greenhouse conditions, disease severity was significantly higher on pyraclostrobin-treated chickpea plants inoculated with QoI-resistant isolates of D. rabiei than sensitive isolates (p-value = 0.001). This indicates that where resistant isolates are located, fungicide failures may be observed in the field. D. rabiei-specific polymerase chain reaction primer sets and hydrolysis probes were developed to efficiently discriminate QoI- sensitive and - resistant isolates.

NEW SINGLE-COPY NUCLEAR GENES FOR USE IN SCALE INSECT SYSTEMATICS

Despite the advent of next-generation sequencing, the polymerase chain reaction (PCR) and Sanger sequencing remain useful tools for molecular identification and systematics. To date, molecular systematics of scale insects has been constrained by the paucity of loci that researchers have been able to amplify with available PCR primers. Due to the rapid molecular evolution of scale insects, “universal” primers, and even primers developed for their sister taxon the Aphidoidea, typically fail. We used transcriptome data for two diaspidids, Acutaspis umbonifera (Newstead) and Chrysomphalus aonidum (Linnaeus), together with a published aphid genome, to design novel PCR primer sets for scale insects. Our primers amplify fragments of eight single-copy genes: ATP-dependent RNA helicase (DHX8), translation initiation factor 5 (IF5X1), DNA replication licensing factor (Mcm2), double-strand break repair protein (MRE11A), serine/threonine- protein phosphatase (PPP1CB), DNA-directed RNA polymerase II (RNApII), ribonucleoside-diphosphate reductase (RRM1), signal recognition particle receptor (SRPα), neuronal PAS domain-containing protein 4 (NPAS4), and cleft lip and palate transmembrane protein 1 (TP1). Here we report the results of tests of amplification success and phylogenetic utility of these primer sets across the Diaspididae and nine other families of Coccomorpha.

감자T바이러스 검정을 위한 RT-PCR 및 Nested PCR 진단시스템 개발

Potato virus T (PVT) is a plant pathogen in the family Betaflexiviridae, group IV single-stranded positive sense RNA viruses. The major host of PVT is potato, and it has been reported in Ullucus tuberosus, Oxalis tuberosa and Tropaeolum tuberosum. This study aimed at developing reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR techniques for specific detection of PVT. Finally, Two RT-PCR primer sets were developed and verified. The RT-PCR products were amplified to 734 (PVT RT-PCR primer set 6) and 828 bp (PVT RT-PCR primer set 29) long to detect PVT. The nested PCR primer sets [PVT-N70/C20 (734→315 bp) and PVT-N75/C30 (828→529 bp)] were developed which are high sensitivity and verification for detection of PVT. Furthermore, a modified-positive control plasmid is use to verify contamination of laboratory in PVT detection. This study supported the diagnose PVT in potato or PVT related hosts.

Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification.

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max). Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction) primer set and seed soaking (without DNA extraction) for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence) and one naturally infected seed in a 300-seed sample (0.33 % incidence). The PCR-based assay was rapid (< 9 h), did not require DNA extraction and was very sensitive.