Real-time PCR Method For Detection Research Articles

Nuclease Enrichment and qPCR Detection of Rare Nucleotide Variants.

The emergence of circulating DNA analysis in blood during the past decade has responded to the need for noninvasive alternatives to classical tissue biopsies. This has coincided with the development of techniques that allow the detection of low-frequency allele variants in clinical samples that typically carry very low amounts of fragmented DNA, such as plasma or FFPE samples. Enrichment of rare variants by nuclease-assisted mutant allele enrichment with overlapping probes (NaME-PrO) enables a more sensitive detection of mutations in tissue biopsy samples alongside standard qPCR detection assays. Such sensitivity is normally achieved by other more complex PCR methods, such as TaqMan qPCR and digital droplet PCR (ddPCR). Here we describe a workflow of mutation-specific nuclease-based enrichment combined with a SYBR Green real-time quantitative PCR detection method that provides comparable results to ddPCR. Using a PIK3CA mutation as an example, this combined workflow enables detection and accurate prediction of initial variant allele fraction in samples with a low mutant allele frequency (<1%) and could be applied flexibly to detect other mutations of interest.

Two species-specific TaqMan-based quantitative polymerase chain reaction assays for the detection in soil of Paenibacillus polymyxa inocula.

The increasingly widespread use of beneficial microbial inocula in agriculture gives rise to two primary needs: i) the assessment of the environmental risk, i.e. their impact on local soil microbiome and soil properties; ii) being able to track them and monitor their persistence and fate to both optimize their formulation and application method. In previous years, PCR-based methods have detected bacterial or fungal bioinoculant at the species or strain level. However, the selective detection, quantification, and monitoring of target microbial species in a complex ecosystem such as soil require that the tests possess high specificity and sensitivity. The work proposes a quantitative real-time PCR detection method using TaqMan chemistry, showing high specificity and sensitivity for the Paenibacillus polymyxa K16 strain. The primer and probe sets were designed using the polymyxin gene cluster targeting pmxC and pmxE sequences. Validation tests showed that these assays allowed a discriminant and specific detection of P. polymyxa K16 in soil. The TaqMan-assay developed could thus ensure the necessary level of discrimination required by commercial and regulatory purposes to detect and monitor the bioinoculant in soil.

A New Approach Using the SYBR Green-Based Real-Time PCR Method for Detection of Soft Rot Pectobacterium odoriferum Associated with Kimchi Cabbage.

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferum- specific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.

Real-time PCRmethod for detection of short DNA using a deoxyuridine probe and application for detection of fomivirsen.

This study sought to develop a short DNA detection method using a deoxyuridine probe and polymerase chain reaction. The probe was hybridized to the target short DNA, which was then extended by DNA polymerase. The extended DNA was used for real-time PCR after the probe was removed by uracil DNA glycosylase. This method measured from 0.01 to 10nM of a model short DNA sequence of 17 nucleotides. The method was then used to detect the nucleic acid medicine fomivirsen, as well as 21 phosphorothioate nucleotides, and to quantify 0.1-100nM of fomivirsen. This method may be useful for detecting short DNA fragments, such as functional nucleotides.

Establishment and Application of a Quantitative PCR Method for E248R Gene of African Swine Fever Virus.

Simple SummaryAfrican swine fever (ASF) is the most serious animal disease that endangers the pig industry in China, causing the heaviest economic loss so far. Effective prevention and control of ASF is necessary for China’s pig industry, and also the focus and hotspot of global swine industry disease prevention and control research. In view of this, rapid, specific and sensitive diagnosis of ASF is of great significance. In this study, ASF virus (ASFV) E248R gene was selected to be the target for establishing a real-time PCR method which did not cross-react with other porcine viruses that could cause similar symptoms. The detection methods can be used for the efficient detection of ASFV infection and the recombinant live-vectored virus-expressing antigen protein of ASFV.ASF has caused huge economic losses to China’s swine industry. As clinical symptoms of ASF were difficult to distinguish from classical swine fever and porcine reproductive and respiratory syndrome (PRRS), rapid and effective differential diagnosis of ASFV seems very important to control the spread of the disease. In this study, the ASFV E248R gene was selected to be the target for establishing a real-time PCR method. TaqMan real-time PCR for the detection of ASFV E248R gene did not cross-react with other porcine viruses that could cause similar symptoms. The results of the repeatability test showed that the coefficients of variation between and within groups were lower than 1.977%. This method can be used for the rapid detection and early diagnosis of ASF. Meanwhile, the recombinant PRRS virus (PRRSV)-expressing E248R gene of ASFV was constructed and rescued by using the reverse genetic platform of live-attenuated PRRSV vaccine. The ASFV E248R gene can be detected by using this real-time PCR detection method, confirming that the ASFV E248R gene could be stably amplified in PRRSV genome at least 20 cell passages. The detection methods can be used for the efficient detection of the ASFV infection and recombinant PRRSV live vector virus-expressing ASFV antigen protein.

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystisplatani, the Agent of Canker Stain of Platanus spp.

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.

Establishment of a Duplex Real Time PCR Method for Detection of SARS-COV-2

To quickly and efficiently detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and prevent and control the spread of novel coronavirus disease (COVID-19), a highly sensitive duplex real-time PCR (RT-PCR) detection method has been established. In this study, the specificity of primers and probes were designed, respectively, according to the ORF1ab gene and N gene sequence of SARS-COV-2, and fluorescent probes were labeled with carboxyl fluorescein (FAM) and green fluorescent protein (VIC). The duplex RT-PCR method for detecting SARS-COV-2 with TaqMan probe was established, which has a limit of detection of 10 copies/µL, and the linear detection range of ORF1ab and N gene were 1.0 × 101-1.0 × 105 copies/µL and 1.0 × 101-1.0 × 106 copies/µL, respectively, realizing the simultaneous detection of ORF1ab and N genes in simulated SARS-COV-2 samples. The method has high sensitivity, accurate quantification, simple operation, and cost-saving, which can be used for rapid and efficient quantitative detection of SARS-COV-2.

Development of a Novel RT-qPCR Detecting Method of Covert Mortality Nodavirus (CMNV) for the National Proficiency Test in Molecular Detection.

Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R2) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.

Development and evaluation of a qPCR detection method for citrinin in Liupao tea

Penicillium is universal in dark tea, and Penicillium citrinum can produce a kidney toxin called citrinin (CIT). Determining CIT is difficult because of the complexity of the dark tea substrate and the diversity of CIT-producing fungi. Therefore, this study established a real-time PCR (qPCR) detection method for CIT-related synthetic genes (ctnD, orf1, ctnA, pksCT, orf5, orf7, and ctnG) in Liupao tea and determined the content of CIT in samples at different production stages and the toxin-producing abilities of fungi (Aspergillus oryzae, etc.) in Liupao tea. CIT was found in all samples during the pile-fermentation process of Liupao tea, and CIT was detected in two samples during the aging process. The established method demonstrated good sensitivity and specificity in detecting CIT-related synthetic genes. The reaction efficiency was within the preferred range of 100 ± 10%. CIT was not detected or was below the detection limit when the Ct value of one or more related synthetic genes was greater than 33.5. Therefore, the established qPCR method can effectively predict the production of CIT in Liupao tea, and it is applicable to the judgment of whether fungi produce CIT.

Establishment of a Dual Real-Time PCR Assay for the Identification of African Swine Fever Virus Genotypes I and II in China.

Since the first outbreak of ASFV genotype II in China in 2018, ASF has posed a significant threat to the swine industry. After the emergence of genotype I in China in 2020, the epidemic prevention and control have become more difficult. No effective commercial vaccine is currently available, and the disease is difficult to eradicate; therefore, the identification of the ASFV genotype is critical to establish biosafety control measures. In this study, a dual real-time PCR detection method based on B646L and E183L genes was developed to distinguish between ASFV genotypes I and II by specifically amplifying the genotype I E183L gene. The method is strongly specific, detects B646L and E183L genes simultaneously, and does not cross-react with PEDV, PCV, PRRSV, PRV, and CSFV. The double real-time PCR detection of ASFV genotypes I and II showed a B646L amplification curve, and only genotype I showed an E183L amplification curve, consistent with our expectations. The method has high sensitivity and the lowest copy numbers detected for recombinant plasmids B646L and E183L were 1.07 × 102 and 3.13 × 104 copies/μL, respectively. The method is reproducible, and the coefficient of variation for detecting the coefficient of variation (CV) values of the two recombinant plasmids was <2%. Seven samples were positive and 277 were negative, and the results of the two methods were consistent. The dual real-time PCR presented in this study provides a rapid detection method for the identification of ASFV genotypes I and II, which may lead to improving efficient prevention and control measures for ASF in China.

Development of a duplex real-time PCR assay for simultaneous detection and differentiation of Theileria equi and Babesia caballi.

Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

Enterocytospora artemiae (EAM) mainly parasitizes the hepatopancreas of Palaemonetes sinensis. Serious infection leads to hepatopancreatic lesions, which greatly reduce the vitality of P. sinensis. Currently, EAM is detected via conventional PCR methods. However, conventional PCR has low sensitivity and cannot be used for accurate quantitative detection of EAM or its parasitic activity in host tissues. In this study, we designed a pair of specific primers based on the sequence of the ribosomal protein S9 gene (RPS9; GenBank accession number: MZ420734) to establish and optimize a SYBR Green I real-time fluorescent quantitative PCR detection method for EAM. Only EAM appeared as a bright and single target band, whereas other microorganisms did not, indicating that the primer for RPS9 had high specificity. This method displayed optimum amplification effects at an annealing temperature of 55°C, and the melting curve of the product produced a single peak. The established method showed a good linear relationship from 2.2 × 108 to 2.2 × 101 copies/μL. The relationship between the number of cycle thresholds (Ct) and the logarithm of the initial template amount (x) conformed to Ct = −3.281 log x + 36.543 (R2 = 0.998). Amplification efficiency was 101.737%, and the lower limit of detection sensitivity was 2.2 × 101 copies/μL. Good intra- and inter-group repeatability was observed within the linear range. The sensitivity of this method was more than 200 times higher than that of nested PCR. Thus, detection data obtained using this method may be useful as a technical reference for rapid and accurate identification of EAM infection and for the prevention and control of EAM during P. sinensis breeding.

Development of a duplex real-time PCR method for the detection of influenza C and D viruses

Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide. Genome structure of influenza D virus (IDV) is identical to that of influenza C virus (ICV), and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology. To date, the prevalence of ICV and IDV in China is unclear, but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential. To efficiently monitor ICV and IDV, it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research. A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed. TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed. This method exhibited good specificity and sensitivity, and the detection limit reached 1 × 101 copies/μL of plasmid standards of each pathogen. Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method. One positive sample of IDV was detected, and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing. The duplex real-time PCR detection method represents a sensitive and specific tool to detect ICV and IDV. It provides technical support for virus research and clinical diagnosis of ICV and IDV. This information will benefit animal and human health.

Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA

Objective To establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). Methods HBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. Results The HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 4.41%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0.038). Conclusion The established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.

Comparison of three real-time PCR methods for detection of macrolide-resistant Mycoplasma genitalium in Sweden

There is a worldwide increase in macrolide-resistant Mycoplasma genitalium strains, with severe impacts on treatment. The aim of this study was to compare three real-time PCR methods for the detection of macrolide resistance: an in-house PCR described by Touati et al., ResistancePlus® MG (SpeeDx), and S-DiaMGRes™ (Diagenode Diagnostics). One hundred M. genitalium-positive patient samples collected in Sweden and a quantitated M. genitalium DNA control were analyzed. Macrolide resistance was detected in 18, 15, and 16 of the samples with the respective methods. Sequencing of the 23S rRNA gene confirmed resistance in 16 (16%) of 100 samples in which it was detected with any of the three methods. ResistancePlus® MG and S-DiaMGRes™ falsely determined one sample as macrolide-sensitive, but this sample was determined as resistant when retested. The sensitivity of the methods was comparable, although there should be awareness of possible incorrect determination of macrolide resistance, especially of low-positive samples.

Development of a Quantitative Real-Time PCR Assay for Detection of Cryptosporidium spp. Infection and Threatening Caused by Cryptosporidium parvum Subtype IIdA19G1 in Diarrhea Calves from Northeastern China.

Parasitic diarrheal disease is a major cause of morbidity and mortality in the developing world. Calves are highly susceptible to Cryptosporidium spp. infection that resulted in diarrhea, growth retardation, and weight loss, and was one of the most common enteropathogens. It is especially difficult for molecular detection of calves with inapparent or subclinical infections of cryptosporidiosis. In view of this, this study established a real-time quantitative PCR (RT-qPCR) detection method to clarify its epidemic characteristics, based on Cryptosporidium 18S rRNA gene with the 150 bp product length to investigate the infection of Cryptosporidium spp. in northeastern China The standard curve equation is Ct = -2.91 × lg (Cryptosporidium spp. copies) +10.18, with better sensitivity, stability, and reproducibility. A total of 148 out of 425 fecal samples (34.82%) were detected Cryptosporidium positive with RT-qPCR, including (36.11%) in Heilongjiang province (29.60%), (29.6%) in Jilin province, and (37.50%) in Liaoning province. The infection prevalence of Cryptosporidium parvum, Cryptosporidium ryanae, Cryptosporidium andersoni, and Cryptosporidium bovis from calves in order from high to low was 14.35% (95% confidence interval [CI], 11.2-18.1), 6.12 (95% CI, 4.0-8.8), 2.35 (95% CI, 1.1-4.3), and 0.47 (95% CI, 0.1-1.7), respectively, suggesting C. parvum was the predominant species in calves in northeastern China. Using 60-kDa glycoprotein gp60 gene, all of the 61 C. parvum-positive specimens were further precisely confirmed to IIdA19G1 subtype. This suggested that IIdA19G1 subtype of C. parvum could threaten to cause diarrhea calves from notheastern China (p < 0.01). The prevalence of 34.82% (148/425) using RT-qPCR had a significant difference compared with the prevalence of nested-PCR (23.29%) and microscopic examination (3.76%). The findings improved the epidemiological knowledge of calves infected with cryptosporidiosis in China, highlighting the importance of ongoing Cryptosporidium surveillance.

A novel quantitative real-time PCR method for the detection of mammalian and poultry species based on a shared single-copy nuclear DNA sequence

Accurate quantification of species fractions is critical to determine meat adulteration. This study aimed to develop a novel quantitative real-time PCR (qRT-PCR) method for detection of mammalian and poultry DNA. A shared single-copy nuclear DNA sequence derived from the first exon of the LcoR gene was identified as a multi-species universal reference for a qRT-PCR assay. The conservation and copy number of the LcoR gene were evaluated among different species. The limit of detection was 0.01 ng DNA or 0.01% meat ingredient, and the limit of quantification was 0.01 ng DNA or 0.05% meat ingredient. Both the relative error (R.E.) and relative standard deviation (R.S.D.) were ≤ 25%. Moreover, modified coefficient k was introduced into this quantitative system to improve the accuracy and reliability of results, with maximum R.E. improved from 19.43% to 16.16%. The quantitative method would contribute to fighting against meat adulteration and maintaining a fair market.

A TaqMan-based quantitative real-time PCR assay for identification of the goose circovirus

Goose circovirus (GoCV) is a potential immunosuppressive virus that poses a great hazard to the goose industry and has been shown to be widely distributed throughout China. We have established a fast, sensitive and highly specific TaqMan real-time quantitative PCR detection method for this virus. Specific primers and probes were designed against the conserved regions of the genomic GoCV Rep gene. The results showed that the assay was highly specific and sensitive for GoCV and did not cross-react with other non-targeted waterfowl viruses. The established method will be helpful for epidemiological detection and may be effective in the prevention and control of the disease.

Improved DNA extraction and quantitative real-time PCR for genotyping Erysiphe necator and detecting the DMI fungicide resistance marker A495T, using single ascocarps

DNA extraction from minute fungal samples is challenging in all genetic studies. Identification of genetic groups and population biology mostly rely on the laborious production of single conidium isolates or on field samples, including infected plant materials. This paper reports a simple and cost-effective protocol for DNA extraction from individual chasmothecia of Erysiphe necator for subsequent applications. It is a less laborious alternative for genotyping purposes than production and analysis of single conidium isolates or analysis of infected plant material from the field. Using the protocols described here for 186 E. necator samples tested, genetic groups A and B were assigned. Based on CYP51 sequences, all the samples belonged to group B, while TUB2 sequences exhibited SNPs also diagnostic for group A. Additionally, a quantitative real-time PCR detection method of single nucleotide polymorphism in the CYP51 gene associated with DMI fungicide resistance was applied. The A495T marker, associated with DMI resistance, and here reported for the first time from Hungary, was detected by quantitative real-time PCR assays and direct sequencing of CYP51. The methods developed in this study can be applied as routine tests to monitor powdery mildew populations for fungicide resistance and other genetic characteristics.

Improved DNA extraction and quantitative real-time PCR for genotyping Erysiphe necator and detecting the DMI fungicide resistance marker A495T, using single ascocarps

DNA extraction from minute fungal samples is challenging in all genetic studies. Identification of genetic groups and population biology mostly rely on the laborious production of single conidium isolates or on field samples, including infected plant materials. This paper reports a simple and cost-effective protocol for DNA extraction from individual chasmothecia of Erysiphe necator for subsequent applications. It is a less laborious alternative for genotyping purposes than production and analysis of single conidium isolates or analysis of infected plant material from the field. Using the protocols described here for 186 E. necator samples tested, genetic groups A and B were assigned. Based on CYP51 sequences, all the samples belonged to group B, while TUB2 sequences exhibited SNPs also diagnostic for group A. Additionally, a quantitative real-time PCR detection method of single nucleotide polymorphism in the CYP51 gene associated with DMI fungicide resistance was applied. The A495T marker, associated with DMI resistance, and here reported for the first time from Hungary, was detected by quantitative real-time PCR assays and direct sequencing of CYP51. The methods developed in this study can be applied as routine tests to monitor powdery mildew populations for fungicide resistance and other genetic characteristics.