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Lactoferrin, a natural antimicrobial compound found in a variety of mucosal surface, is a potential candidate gene in cattle for increasing resistance to various infectious diseases. Previously reported A/C polymorphism (-28 A/C) within TATA box sequence of lactoferrin ( LTF) gene was shown to be a key factor in lactoferrin expression level. The objective of this study was to develop an allele-specific PCR method for high-throughput screening of single nucleotide polymorphisms at lactoferrin TATA box in dairy cattle. The technique involves PCR amplification of specific alleles that differ only in one base. This approach, which was validated on three known TATA box genotypes, is accurate, sensitive, and suitable for high-throughput screening since it requires only one reaction per case; it is performed in a single tube and also it needs minimal sample processing. [ABSTRACT FROM AUTHOR]

NADP-dependent enzyme isocitrate dehydrogenase ( IDH) mutations, IDH1 and IDH2, have been described in acute myeloid leukemia (AML) using next generation sequencing approaches. IDH2 mutations are heterozygous; they alter a single arginine residue at position 140 or 172 and have distinct prognostic significance. The current detection methods of IDH2 mutations are laborious and time consuming as they require DNA sequencing. Herein, we report a new allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) method to detect the IDH2 mutations. Analysis of leukemic DNA samples from 120 AML patients enabled to identify IDH2 mutations in 22 cases which were confirmed by direct DNA sequencing. Of these, 17 harbored IDH2 (R140Q) and 5 IDH2 (R172K) mutations. Serial dilution experiments showed that the assay enable to detect mutations in 10 dilutions. Our ASO-PCR method appears useful for routine diagnostic screening of these prognostically relevant alterations in AML and may be conveniently included in the diagnostic workup. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancers (CRCs) with wild-type KRAS respond to EGFR-targeted antibody treatment. Analysis of the hotspot clustered mutations in codons 12 and 13 is compulsory before therapy and no standardized methodology for that purpose has been established so far. Since these mutations may have different biological effects and clinical outcome, reliable frequency and types of KRAS mutations need to be determined for individual therapy. Aims: The purpose of this study was to describe the KRAS mutation spectrum in a group of 481 Serbian mCRC patients and to compare the general performances of allele-specific PCR and reverse-hybridization assays. Methods: KRAS testing was performed with two diagnostic analyses, DxS TheraScreen K-RAS PCR Kit and KRAS StripAssay™. Results: KRAS mutations in codons 12 and 13 were present in 37.6 % of analyzed formalin-fixed paraffin-embedded (FFPE) DNA samples. The seven most frequent mutation types were observed with both assays: p.G12D 34.6 %, p.G12V 24.9 %, p.G12A 10.3 %, p.G12C 8.1 %, p.G12S 5.4 %, p.G12R 1.6 %, and p.G13D 15.1 %. Regarding double mutants, 0.8 % of them were present among all tested samples and 2.2 % among KRAS mutated ones. Conclusions: Two screening approaches that were used in this study have been shown as suitable tests for detecting KRAS mutations in diagnostic settings. In addition, they appear to be good alternatives to methods presently in use. In our experience, both methods showed capacity to detect and identify double mutations which may be important for potential further subgrouping of CRC patients. [ABSTRACT FROM AUTHOR]

Mutations of components of the mitogen-activated protein kinase pathway, mainly BRAF, are common in serous ovarian borderline tumors, whereas high-grade serous ovarian carcinomas rarely show this feature. With the advent of specific kinase inhibitors active against BRAF-mutated cancers, rapid and sensitive detection of the BRAF V600E, by far the most common mutation of this gene, is of great practical relevance. Currently, BRAF mutations are detected by DNA-based techniques. Recently, a monoclonal antibody (VE1) specific for the BRAF V600E protein suitable for archival tissues has been described. In this study, we compared detection of the V600E mutation in serous ovarian tumors by VE1 immunostaining and by allele-specific polymerase chain reaction. All 141 cases of high-grade serous ovarian cancer showed negative or rarely weak, diffuse background VE1 immunostaining, and BRAF wild type was confirmed by molecular analysis in all tested cases. In contrast, 1 (14%) of 7 low-grade serous carcinomas and 22 (71%) of 31 serous borderline tumors revealed moderate to strong VE1 positivity. Immunostaining was clearly evaluable in all cases with sufficient tumor cells, and only rare cases with narrow cytoplasm were difficult to interpret. The V600E mutation was confirmed by allele-specific polymerase chain reaction and sequencing in all VE1-positive cases. Two VE1-positive cases with low epithelial cell content required repeat microdissection to confirm the presence of the mutation. Immunohistochemistry with the VE1 antibody is a specific and sensitive tool for detection of the BRAF V600E mutation in serous ovarian tumors and may provide a practical screening test, especially in tumor samples with low epithelial content. [ABSTRACT FROM AUTHOR]

Rhubarb (official Da-huang) is an important medicinal herb in Asia. Many adulterants of official Da-huang have been discovered in Chinese markets in recent years, which has resulted in adverse effects in medicinal treatment. Here, novel molecular markers based on a short maturase K ( matK) gene were developed for authenticating official Da-huang. This study showed that all the species from official Da-huang were clustered together in one clade in the polygenetic trees based on short matK. Two highly conserved single nucleotide polymorphisms of short matK were mined in the species from official Da-huang. Based on these polymophisms, four improved specific primers of official Da-huang were successfully developed that generated reproducible specific bands. These results suggest that the short matK sequence can be considered as a favorable candidate for distinguishing official Da-huang from its adulterants. The established multiplex allele-specific PCR was determined to be simple and accurate and may serve as a preferable tool for authentication of official Da-huang. In addition, we suggest that short-sized specific bands be developed to authenticate materials used in traditional Chinese medicine. [ABSTRACT FROM AUTHOR]

. Hepatitis B virus (HBV) pol/RT mutations that confer clinical resistance to tenofovir disoproxil fumarate (TDF) have not been detected to date. In vitro, the rtN236T adefovir dipivoxil (ADV)-associated resistance mutation confers low-level cross-resistance to tenofovir: 3- to 13-fold changes in EC50 from wild type. This study evaluated the clinical response of rtN236T mutant viruses by comparing their early viral load decay kinetics to wild-type viruses in chronic HBV monoinfected patients harbouring rtN236T prior to initiating TDF or emtricitabine (FTC)/TDF therapy. Baseline samples ( n = 105) from adefovir refractory patients were tested for the presence of rtN236T using a highly sensitive allele-specific PCR assay with an rtN236T detection cut-off of 0.5%. The rtN236T mutation was detected at baseline in 14.3% (14/98) of analysable patient samples (0.5-93.2%, rtN236T percentage range). The median change in total HBV DNA at week 24 was comparable for patients with rtN236T detected at baseline (−3.7 log10 copies/mL, n = 14) as compared to patients with wild-type HBV (−3.2 log10 copies/mL, n = 90). In patients with rtN236T, wild-type and rtN236T mutant virus showed similar rates of HBV DNA decline with no statistically significant difference observed at week 4. Moreover, the proportion of rtN236T remained unchanged in patients in either arm of the study during treatment. In conclusion, the rtN236T mutant virus showed similar HBV DNA decline kinetics to wild-type virus in adefovir refractory patients who switched to TDF or FTC/TDF. Despite low levels of cross-resistance in vitro, TDF similarly suppresses wild-type and rtN236T mutant viruses in vivo. [ABSTRACT FROM AUTHOR]

El artículo discurre sobre la bacteria bodetella pertussis, la toxina pertúsica y los alelos relacionados. Los autores comentan sobre las vacunas anti-pertúsicas, la regulación genética y los antígenos utilizados para inducir inmunidad. Otros temas incluyen las reacciones en cadena de la polimerasa (PCR) y una PCR alelo específica.

Abstract: The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Brazil and other countries. Its control is becoming more difficult due to the development of resistance in populations. Early detection of resistance can help in developing effective control strategies. This study evaluated the susceptibility of R. microplus to cypermethrin and chlorpyriphos and was the first attempt to identify the mechanism of resistance (target site insensitivity) in cattle tick populations from Minas Gerais state (Southeastern Brazil). Engorged female ticks were collected from 10 ranches within the state of Minas Gerais, and susceptibility was evaluated with the larval packet test (LPT) using technical grade cypermethrin and chlorpyriphos. It was possible to analyze LPT results of seven populations. Target site insensitivity was investigated in all 10 isolates by using molecular approaches for detection of the T2134A substitution within the domain III S6 segment and the C190A in the domain II S4-5 linker from the para-type sodium channel gene. LPT showed that all seven populations were resistant to cypermethrin with resistance ratio (RR) ranging from 16.0 to 25.0 and 85.7% were resistant to chlorpyriphos (RR=2.2–15.6). Although the T2134A mutation was not detected, the C190A mutation was highly prevalent, being present in 82–100% of the alleles sampled in field populations. A significant correlation was found between the LC50 values for cypermethrin and the frequency of the C190A mutation suggesting that it might be responsible for the phenotypic resistance detected. [Copyright &y& Elsevier]

ABSTRACT In the present study, an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 5 (TLR5) (C1205T; P402L) that is related to the impaired recognition of Salmonella enterica serovar Choleraesuis (SC) was developed. The allele frequencies in several pig breeds in Japan and the Czech Republic were also compared. The swine TLR5 C1205T mutation was successfully determined by ASP-PCR using genomic DNA samples in Japan that had previously been genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from blood obtained from 110 pigs from seven different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR completely matched the results from the sequencing method. The allele frequency of the swine TLR5 C1205T mutation was 27.5% in the Landrace breed of the Czech Republic compared with 50.0% in Japanese Landrace. In Japan, the C1205T mutation was found only in the Landrace breed, whereas in the Czech Republic it was found in both the Landrace and Piétrain breeds. These results indicate the usefulness of ASP-PCR for detecting a specific SNP for swine TLR5 affecting ligand recognition. They also suggest the possibility of genetically improving pigs to enhance their resistance against SC infection by eliminating or selecting this specific SNP of swine TLR5. [ABSTRACT FROM AUTHOR]

The article discusses a study which developed an allele-specific primer-polymerase chain reaction (ASP-PCR) designed for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G). Study authors compared the allele frequency among pig breeds of Japan and the Czech Republic, and found that C406G mutation is only present in the Landrace breed in Japan and the Landrace breed in the Czech Republic.

The rDNA ITS region of 18 samples of Changium smyrnioides from 7 areas and of 2 samples of Chuanminshen violaceum were sequenced and analyzed. The amplified ITS region of the samples, including a partial sequence of ITS1 and complete sequences of 5.8S and ITS2, had a total length of 555 bp. After complete alignment, there were 49 variable sites, of which 45 were informative, when gaps were treated as missing data. Samples of C. smyrnioides from different locations could be identified exactly based on the variable sites. The maximum parsimony (MP) and neighbor joining (NJ) tree constructed from the ITS sequences based on Kumar's two-parameter model showed that the genetic distances of the C. smyrnioides samples from different locations were not always related to their geographical distances. A specific primer set for Allele-specific PCR authentication of C. violaceum from Jurong of Jiangsu was designed based on the SNP in the ITS sequence alignment. C. violaceum from the major genuine producing area in Jurong of Jiangsu could be identified exactly and quickly by Allele-specific PCR. [ABSTRACT FROM AUTHOR]

Background & objectives: Genetic diagnosis of spinal muscular atrophy (SMA) is complicated by the presence of SMN2 gene as majority of SMA patients show absence or deletion of SMN1 gene. PCR may amplify both the genes non selectively in presence of high amount of DNA. We evaluated whether allele-specific PCR for diagnostic screening of SMA is reliable in the presence of high amount of genomic DNA, which is commonly used when performing diagnostic screening using restriction enzymes. Methods: A total of 126 blood DNA samples were tested in amounts ranging 80-200 ng, referred for the genetic diagnosis of SMA using both conventional PCR-RFLP and allele-specific PCR. Results: The results from both methods showed agreement. Further, allele-specific PCR was found to be a time-efficient and cost-effective method. Interpretation & conclusions: Our study demonstrated the accuracy of our allele-specific PCR and the results were comparable compatible with that of PCR-RFLP, indicating its practical application in SMA diagnostic screening. [ABSTRACT FROM AUTHOR]

Background: Bovine tuberculosis is a chronic bacterial disease of cattle that occasionally affects human. A unique mutation at position 169 (C→G) in Mycobacterium bovis differentiates it from Mycobacterium tuberculosis. For the first time, PCR-RFLP method was used in this study to separate the two organisms. Methods: In this study, 98 tuberculosis patients were assessed using microscopic evaluations, culture and antibiogram. Then, nucleotide changes in position 169 were investigated using Allele-specific PCR and PCR-RFLP methods. Finally, the results were compared with spoligotyping results . Findings: Antibiogram revealed 28 subjects (28.57%) to be resistant and 54 (55.1%) to be sensitive to pyrazinamide while and 16 cases (16.23%) showed no growth. Of 28 resistant patients, 3 (3.1%) were diagnosed with M. Bovis using the classical methods. Using molecular methods, 95 (96.93%) out of 98 subjects were diagnosed with M. tuberculosis and 3 (3.06%) with M. bovis. Allele-specific PCR and PCR-RFLP matched with spoligotyping results. Conclusion: Our result showed the incidence of M. bovis infection to be higher in Iranian population (3.1%) than other studied cases (0.5-1%). In addition, we showed that Allele-specific PCR is the method of choice because it is faster and cheaper than PCR-RFLP. However, PCR-RFLP could be a proper alternative to prevent probable mismatches in Allele-specific PCR. [ABSTRACT FROM AUTHOR]

Abstract: Background: The CDA 79A>C (K27Q, rs2072671) functional SNP has recently shown a crucial role in the pharmacogenetics of cytidine-based anticancer drugs widely administered to different subsets of patients. Current gold standard in screening for the CDA rs2072671 is the sequence-based genotyping method. Here we developed a novel, rapid Allele-Specific PCR method for CDA rs2072671 genotyping. Methods: DNA was extracted from 324 healthy individuals from two different populations (Italian and Han Chinese). CDA rs2072671 genotyping was performed by Allele-Specific PCR. Sequencing was performed to validate the test results. Results obtained from population screening were compared to that already available in HapMap and in the literature. Results: Samples analyzed were successfully genotyped and the results were confirmed by sequencing. Genotype distribution does not differ significantly from that previously reported for each relative ethnic group. Also, the world-wide distribution of the CDA rs2072671 SNP is reported. A striking difference is present among the main ethnicities (p=1.715×10−77), with CDA*27Q allele showing the lowest frequency in African group (9.7%) and the highest in Caucasians (35.9%). Conclusion: This Allele-Specific PCR method is a useful tool in pharmacogenetics research and a valid and reliable alternative for CDA rs2072671 screening where sequencing or Real-Time PCR is not available. [Copyright &y& Elsevier]

A compatible individual-identification system with the multiplex SNuPE assay system, which was constructed of eighteen highly polymorphic and codominant single nucleotide polymorphisms (SNPs), was developed in the Japanese black pine (Pinus thunbergii Parl.). The eighteen SNPs were transformed into dominant markers by employing an allele-specific PCR (ASP) assay. Three multiplex PCR sets, each containing six ASP markers, were designed to increase cost saving and assay simplicity. Analyses of statistical parameters of 18 ASP markers revealed that gene diversity (H) and polymorphic information contents (PIC) were 0.331 to 0.500 (average of 0.425), and 0.375 to 0.500 (average of 0.473), respectively. The cumulative confusion probability (Ccumulative) of the system was extremely low (3.14 x 10-6), roughly one chance in 318,000 genotypes. The cumulative discrimination power (Dcumulative) was exceedingly high (0.999997), which means that the identification system is significantly powerful to discriminate the unrelated individuals. [ABSTRACT FROM AUTHOR]

Abstract: In an attempt to insert the modified castor bean catalase intron (mCBC intron) into the coding sequence of the Cre recombinase gene, we found that the mCBC intron was not completely spliced from the resulting iCre gene in tobacco and Arabidopsis. Sequencing and allele-specific PCR analyses indicated that six nucleotides (UUACAG) at the 3′ terminus of the mCBC intron were retained in the mature mRNA of the iCre gene. Moreover, the mCBC intron was incompletely spliced from the Gus gene in pCAMBIA vectors. A mutational analysis of the mCBC intron demonstrated that the incomplete splicing was due to an artificial 3′ splice site introduced by the insertion of an adenine, which created a TAG (stop) codon near the 3′ splice site of the original CBC intron. Deletion of the inserted adenine or the six nucleotides that were retained from the mCBC intron led to the complete removal of the intron from the resulting iCre2 and iCre3 genes. Thus, in this study, we not only characterized the incomplete splicing event of the mCBC intron in tobacco and Arabidopsis, but also reported the construction of two intron-containing Cre recombinase genes that are useful for plant biotechnology applications. [Copyright &y& Elsevier]

Abstract: The study was designed to compare the in vitro egg hatch test (EHT) and molecular allele-specific polymerase chain reaction PCR (AS-PCR) methods as tools for detection of benzimidazole resistance in Haemonchus contortus, a nematode parasite of small ruminants. Comparisons were made during the course of an experimental infection and changes in EHT and AS-PCR were monitored to measure the correlation between in vitro and molecular tests. Both methods were carried out according to World Association for the Advancement of Veterinary Parasitology (WAAVP) recommendations. The molecular test was used to discriminate TAC/TTC polymorphism in the beta-tubulin 200 codon of eight (four resistant and four susceptible) isolates of H. contortus. Using DNA from 100 third-stage larvae, the polymerase chain reaction (PCR)-based analysis revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals in all resistant isolates. Both methods showed comparable and reliable results with regard to detection of benzimidazole resistance. The molecular test has an advantage over the EHT because of its higher sensitivity. On the other hand, EHT is less time-consuming, allows reliable detection of <10% resistance allele frequency and is fairly reliable for the detection of benzimidazole resistance under field conditions. [ABSTRACT FROM AUTHOR]

Hemoglobin Constant Spring (HbCS) is the most common nondeletional α-thalassemia variant causing HbH disease, making its detection crucial in populations at risk. Universal newborn screening for HbH is carried out in California. Identification of α-thalassemia genotypes responsible for HbH and HbH-CS requires rapid, accurate and cost-effective genotyping methods suitable for population screening. We incorporated the HbCS mutation into our existing seven-plex genotyping assay for common α-thalassemia deletions. To assess the feasibility and diagnostic utility of this expanded multiplex gap-PCR assay, we determined genotypic frequencies of HbCS in samples referred for α-thalassemia testing between 1 January 2006 and 31 December 2008. During the 3-year study period, 1436 samples were genotyped for α-thalassemia. HbH-CS accounted for 23 (13%) of the 176 cases of HbH disease identified. In a subset of 145 newborns referred by the California NBS program with an elevated Hb Bart’s level at birth, HbH disease was confirmed in 134 (93%) and HbH-CS identified in 13 (10%) of these. This expanded genotyping assay has proven to be a rapid, reliable and clinically useful diagnostic tool for the detection of HbH-CS disease. [ABSTRACT FROM AUTHOR]

Clin Microbiol Infect 2010; 16: 848–851 Non-nucleoside reverse transcriptase inhibitor (NNRTI) therapy failed in 30 patients with the typical human immunodeficiency virus type 1 reverse transcriptase K103N mutation, detected using standard genotyping. Following discontinuation of NNRTI therapy for a median of 55.9 weeks and a decrease of K103N mutant species to undetectable levels in plasma RNA, minority K103N species remained detectable, by allele-specific PCR, for longer periods of time and at higher frequency, in peripheral blood mononuclear cell (PBMC) DNA than in plasma RNA (76.7% and 46.7% of samples with residual K103N species detected at median frequencies of 18.0% and 3.8%, respectively). Analysis of PBMC DNA should be considered when searching for residual K103N mutant species in patients previously exposed to NNRTIs. [ABSTRACT FROM AUTHOR]

Resolution of the two haplotypes present in an individual that is heterozygous at a locus has been a difficult problem for nucleotide sequence-based population genetic studies. Here, we demonstrate a method in which allele-specific polymerase chain reaction (AS-PCR) and computational phasing are combined for relatively high-throughput, efficient resolution of phase in resequencing studies. Using data from multiple loci that were fully experimentally phased, we demonstrate that the popular computational tool PHASE can accurately phase heterozygous individuals with common SNPs (single nucleotide polymorphisms) and/or common haplotypes. However, we also demonstrate that experimental phasing with AS-PCR can efficiently supplement computational phasing, providing a rapid means to phase individuals with rare SNPs or haplotypes and with heterozygous insertion/deletion polymorphisms. By following simple stepwise procedures, AS-PCR can result in much more efficient and accurate experimental phasing of haplotypes than is possible with traditional methods such as cloning. [ABSTRACT FROM AUTHOR]

Abstract: Allele-specific PCR based on subtype consensus sequences is a powerful technique for detecting low frequency drug resistant mutants in HIV-1 infected patients. However, this approach can be limited by genetic variation in the region complementary to the primers, leading to variability in allele detection. The goals of this study were to quantify this effect and then to improve assay performance. [Copyright &y& Elsevier]

Abstract: Allele-specific PCR (AS-PCR) has been widely used for the detection of single nucleotide polymorphism. But there are some challenges in using AS-PCR for specifically detecting DNA variations with short deletions or insertions. The challenges are associated with designing selective allele-specific primers as well as the specificity of AS-PCR in distinguishing some types of single base-pair mismatches. In order to address such problems and enhance the applicability of AS-PCR, a general primer design method was developed to create a multiple base-pair mismatch between the primer 3′-terminus and the template DNA. This approach can destabilize the primer–template complex more efficiently than does a single base-pair mismatch, and can dramatically increase the specificity of AS-PCR. As a proof-of-principle demonstration, the method of primer design was applied in colony PCR for identifying plasmid DNA deletion or insertion mutants after site-directed mutagenesis. As anticipated, multiple base-pair mismatches achieved much more specific PCR amplification than single base-pair mismatches. Therefore, with the proposed primer design method, the detection of short nucleotide deletion and insertion mutations becomes simple, accurate and more reliable. [Copyright &y& Elsevier]

Abstract: Monitoring antiviral resistance in influenza is critical to public health epidemiology and pandemic preparedness activities. Effective monitoring requires methods to detect low-level resistance and to monitor the change in resistance as a function of time and drug treatment. Resistance-conferring single-nucleotide mutations in influenza virus are ideal targets for such methods. In the present study, fives sets of paired TaqMan® allele-specific PCR (ASPCR) assays were developed and validated for quantitative single-nucleotide polymorphism (SNP) analysis. This novel method using ΔCt is termed allele-specific mixture analysis (ASMA) or FluASMA. The FluASMA assays target L26F, V27A, A30T, and S31N mutations in the A/Albany/1/98 (H3N2) M2 gene and H275Y mutation in the A/New Caledonia/20/99 (H1N1) NA gene and have a limit of quantification of 0.25–0.50% mutant. The error for % mutant estimation was less than 10% in all FluASMA assays, with intra-run ΔCt coefficient of variance (CoV) at ≤2% and inter-run ΔCt CoV at ≤5%. Results from the current study demonstrate that FluASMA is a highly sensitive and quantitative SNP analysis method, even for minor mutant components (<1%). [Copyright &y& Elsevier]

Abstract: Antiretroviral drug resistance is a fundamental survival strategy for the virus that stems from its vast capacity to generate diversity. With the recent availability of new ARV drugs and classes, it is now possible to prescribe fully active ART to most HIV-infected subjects and achieve viral suppression even in those with multidrug-resistant HIV. It is uncertain, however, if this scenario will endure. Given that ART must be given for life, and new compounds other than second-generation integrase inhibitors may not reach the clinic soon, all efforts must be done to avoid the development of resistance to the new agents. Here, we discuss relevant aspects for the clinical management of antiretroviral drug resistance, leaving detailed explanations of mechanisms and mutation patterns to other articles in this issue. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010. [Copyright &y& Elsevier]

In this study, we attempted to elucidate the two resistance factors conferring resistance to organophosphates within the Katsunuma population of Drosophila melanogaster (Meigen), one of which has been mapped on the second chromosome and the other on the third chromosome. With regard to the second chromosome factor, we tested susceptibility to malathion of 54 recombinant inbred lines with recombination between ltd and vg. Analyses of variance (ANOVAs) showed highly significant variation in susceptibility to malathion between recombinant lines. In addition, susceptibility of the second-chromosome resistant line to malathion was increased with additional application of piperonyl butoxide, suggesting a member of the Cyp gene family located between ltd and vg. With regard to the third-chromosome factor, we conducted inhibition assays of acetylcholinesterase (AChE) with respect to fenitroxon and carbaryl, to evaluate the contribution of mutated AChE to organophosphate resistance within the Katsunuma population. I50 values of resistant lines, isolated from this population, were about 15 times higher for fenitroxon, and about two times higher for carbaryl, than those of susceptible lines, suggesting the contribution of mutated AChE to organophosphate resistance within the Katsunuma population. We further investigated the genetic variation in the acetylcholinesterase ( Ace) gene within the newly collected Katsunuma population, by using the allele-specific polymerase chain reaction (PCR) approach, and revealed that within this population there were high frequencies of resistant-type mutations at three sites in the Ace gene, which play critical roles in altering sensitivity of AChE to organophosphate and carbamate insecticides. [ABSTRACT FROM AUTHOR]

The article reports on the study conducted to survey M. oryzae isolates from rice in Japan for resistance to Qol fungicides. It includes the test conducted on 813 single-spore isolates of M. oryzae through the use of a mycelial growth test on PDA. Result of the study founds no resistance of M. oryzae to azoxystrobin among 700 isolates tested.

A multiplex allele-specific PCR analysis was developed to identify six "common" genotypes: AA, AO, BB, BO, OO, and AB. This project included a pre-laboratory exercise that provided active learning experiences and developed critical thinking skills. This laboratory resulted in many successful analyses, which were verified by student knowledge of their phenotypes. However, the design was found to be deficient for the analysis of variants. The limitations in the original allied-health multiplex design were verified through a student designed problem-based laboratory project in an advanced level biochemistry class. Variants were further analyzed in an undergraduate research project using SSCP analyses. The topic of ABO genotyping provides several opportunities for student-centered explorations. [ABSTRACT FROM AUTHOR]

Sexual self-incompatibility in European pear ( Pyrus communis L.) is controlled by a single locus ( S-locus) encoding a polymorphic stylar ribonuclease ( S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, genomic DNA sequences corresponding to five new S-RNase alleles (named S 20 , S 21 , S 22 , S 23 , and S 24 ) and to S m were characterized in European pear cultivars. Re-sequencing S q from ‘General Le Clerc’ showed this S-RNase to encode the same protein as S 12 . Based on these findings, a polymerase chain reaction (PCR)-based method was developed for the molecular typing of cultivars bearing 20 S-RNases ( S 1 – S 14 , S m , and S 20 – S 24 ) using consensus and allele-specific primers. Genomic PCR with consensus primers amplified product sizes characteristic of the S-RNases S 1 , S 2 , S 4 , S 10 , S 13 , and S 20 . However, the allele groups S 3 / S 12 , S 6 / S 8 / S 11 / S 22 and S 5 / S 7 / S 9 / S 14 / S m / S 21 / S 23 / S 24 amplified PCR products of similar size. To discriminate between alleles within these groups, primers to specifically amplify each S-RNase were developed. Application of this approach in 19 cultivars with published S-alleles allowed re-evaluation of one of the alleles of ‘Passe Crassane,’ ‘Conference,’ and ‘Condo.’ Finally, this method was used to assign S-genotypes to 37 cultivars. Test crosses confirmed molecular results. [ABSTRACT FROM AUTHOR]

Abstract: Sixty-five isolates of Pencillium digitatum (Pers.:Fr) Sacc., a causative agent of green mold of postharvest citrus, were collected from various locations in Zhejiang province in 2000, 2005 and 2006, and assayed for their sensitivity to the quinone outside inhibitor (QoI) fungicide azoxystrobin. The results showed that azoxystrobin is highly effective against P. digitatum, in vitro, and that the effective concentrations resulting in reduction of conidial germination and mycelial growth by 50% (EC50) averaged 0.0426 µg/ml and 0.0250 µg/ml, respectively. Twenty-eight azoxystrobin-resistant mutants were obtained by UV mutagenesis and subsequent selection on medium amended with azoxystrobin (12 µg/ml) and salicylhydroxamic acid. All obtained mutants were highly resistant to azoxystrobin and their resistance was genetically stable. Analysis of the cytochrome b gene structure of P. digitatum (Pdcyt b) showed the absence of type I intron in the first hot spot region of mutation. These results indicate that P. digitatum is likely to evolve high levels of resistance to azoxystrobin after its application. Analysis of partial sequences of Pdcyt b from both the azoxystrobin-sensitive parental isolate and the 28 azoxystrobin-resistant mutants revealed that a point mutation, which leads to the substitution at code 143 of alanine for glycine (G143A), is responsible for the observed azoxystrobin resistance in the laboratory mutants. Based on this point mutation, two allele-specific PCR primers were designed and optimized for allele-specific PCR detection of azoxystrobin-resistant isolates of P. digitatum. [Copyright &y& Elsevier]

Abstract: Sensitivity of 184 Botrytis cinerea field isolates to a Qo inhibitor (QoI) fungicide azoxystrobin was determined in this study. Among the 184 isolates, seven showed resistance to azoxystrobin. All these seven azoxystrobin-resistant (AR) isolates were also resistant to a benzimidazole fungicide carbendazim, and a dicarboximide fungicide iprodione. Negative cross-resistance between azoxystrobin and a carboxamide fungicide boscalid was not observed in AR isolates of B. cinerea. Analysis structure of partial cytochrome b (cyt b) gene from B. cinerea showed that a 1204-bp intron inserted directly after the codon 143 of cyt b gene was detected from 79 out of 184 isolates, and all the isolates having this intron were sensitive to azoxystrobin. This intron was not detected from any AR isolate. Sequence analysis of partial cyt b gene showed that all AR isolates had a point mutation at codon 143, which caused a change of glycine to alanine (G143A) at this codon position. Based on the point mutation, an allele-specific PCR assay was developed for the rapid detection of AR isolates of B. cinerea. [Copyright &y& Elsevier]

The article focuses on the G143A point mutation in the fungal mitochondrial cytochrome b gene in French Venturia inaequalis. The authors explain that the mutation contributes to the high level of resistance to Qol fungicides. Researchers use polymerase chain reaction (PCR) which detected the Qol-resistant cytochrome b allele either following the cleavage of the amplified market by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. They discovered the efficiency of PCR-based methods in determining the presence of the mutation in the plant.

Abstract: Objective: To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo+ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods: We used the mtDNA 10400 locus to design unmodified and 3′ phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3′ exonuclease activities. The effects of these primers on primer-extension were evaluated by agarose gel electrophoresis. Results: The unmodified primers were extended by both exo− and exo+ polymerase irrespective of whether the primers were matched or mismatched with the templates. However, the 3′ phosphorothioate-modified primers with a terminal mismatch triggered an, “off-switch” of exo+ polymerase when compared to exo−polymerase. Conclusion: The, “on/off” switch constituted by the combination of 3′ phosphorothioate-modified primers with exo+ polymerase is a cost-effective, high-throughput and reliable method for SNP typing, which will be of enormous application in association studies by single nucleotide polymorphism screening. [Copyright &y& Elsevier]

Identifying the Fusarium species cause Fusarium head blight (FHB) and produces mycotoxins in wheat and other cereal is difficult and time consuming because of confusing phenotypic classification systems. In Japan, the F. graminearum complex, F. culmorum, F. avenaceum, and Microdochium nivale predominantly cause FHB. The internal transcribed spacer (ITS) and 5.8S of rDNA, a partial sequence of β-tubulin and mitochondrial cytochrome b ( cytb) genes of the four species were PCR-amplified and analyzed. On the basis of the ITS, β-tubulin and cytb sequences, F. avenaceum and M. nivale are distinct from the F. graminearum complex and F. culmorum, whereas the F. graminearum complex is closely related to F. culmorum. Moreover, thiophanate–methyl-resistant isolates of the F. graminearum complex and F. culmorum did not have an amino acid substitution at amino acid codon 198 or 200 of β-tubulin. In contrast, very highly or highly thiophanate–methyl-resistant isolates of M. nivale had Glu (GAG) substituted with Ala (GCG) or Lys (AAG) at codon 198, respectively. The allele-specific PCR assay was used to identify the F. graminearum complex and F. culmorum, and these Fusarium species could be distinguished rapidly. [ABSTRACT FROM AUTHOR]

The article analyzes dynamic shifts in the HIV-1 quasispecies to estimate the in vivo fitness cost of enfuvirtide resistance. According to the authors, the HIV-1 quasispecies undergoes dynamic changes in response to withdrawal and reinitiation of fusion inhibitor therapy. They add that the relative stability of plasma HIV-1 titers during decay of V38A suggests that factors other than viral fitness likely define viral load set-point in patients with advanced disease.

Abstract: We have developed a novel method for typing single-nucleotide polymorphisms (SNPs) that can be applicable to rapid screening. The method involves the fusion of two PCR techniques, allele-specific PCR (AS-PCR) and l-DNA-tagged PCR (LT-PCR), which enables us to label PCR products with sequence-defined tags of mirror-image DNA (l-DNA). PCR products were applied without any purification or denaturation steps to gold surfaces where complementary single-stranded l-DNA was immobilized, and the products were detected with surface plasmon resonance (SPR) imaging. We were able to clearly discriminate 3 genotypes at position 2677 of the MDR1 gene (G/G-homozygote, G/T-heterozygote, and T/T-homozygote) by comparing SPR difference images. [Copyright &y& Elsevier]

Two genetic variants of the bovine β-casein gene (A1 and B) encode a histidine residue at codon 67, resulting in potential liberation of a bioactive peptide, β-casomorphin, upon digestion. An allele-specific PCR (AS-PCR) was evaluated to distinguish between the β-casomorphin-releasing variants (A1 and B) and the non-releasing variants. AS-PCR successfully distinguished β-casein variants in 41 of 42 animals as confirmed by sequence analysis. Overall, while the incidence of the homozygous A1 and B animals (i.e., homozygous for the histidine residue; 21.4%) was lower than that for animals without the histidine residue (30.9% respectively), 69% of animals carried at least one allele for the histidine residue at codon 67. [ABSTRACT FROM AUTHOR]

Abstract: In recent years, highly sensitive assays have been developed that detect HIV-1 drug resistance mutations when present at less than 1% of the viral population. These assays are powerful tools when attempting to determine the clinical implications of these low level resistant virions after the administration of single-dose nevirapine. This report demonstrates that non-drug resistant polymorphisms in the primer-binding site for the allele-specific PCR (ASPCR) assay impact primer binding resulting in significant discrepancies in the assay''s performance. Specifically, the use of a “universal” set of ASPCR primers caused an overestimation of the K103N (ntAAC) mutation at position 103 of reverse transcriptase when primer binding site polymorphisms resided close to the 3′ end of the allele-specific primer. Drug resistance was predicted at values ranging from 0.69% to 7.69% for a sample containing only 1% resistance mutations and 3.35–31.84% for a sample containing 5% mutations. Conversely, the use of polymorphism-specific primers detected 1.15–1.36% and 5.20–5.71% resistance for the same 1% and 5% samples. The results demonstrate the need to account for sequence polymorphisms when designing and implementing this highly specific assay. [Copyright &y& Elsevier]

Outcrossing or cross hybridization is a potential concern in herbicide-resistant crop management strategies such as in the Clearfield™ rice system. Recent studies have shown that the mutated acetolactate synthase ( ALS) gene that confers resistance to imazethapyr (Newpath) herbicide can be transferred from Clearfield rice cultivars via cross pollination under field conditions to weedy red rice. Resistance of commercial Clearfield rice cultivars to imazethapyr is due to the presence of two point mutations in the ALS gene that result in amino acid substitutions from serine to asparagine (S to D) and glycine to glutamic acid (G to E). We report here development of a DNA-based method that involves application of allele-specific PCR assays to distinguish herbicide-susceptible and resistant ALS alleles in either homozygous or heterozygous genotypes produced from natural outcrosses between Clearfield varieties CL121, CL141, CL161 and weedy red rice. PCR assays that can distinguish between the homozygous and heterozygous imazethapyr-resistant S653D and G654E SNP alleles of the rice ALS gene were developed and evaluated. A total of 483 individual red rice plants were successfully screened for the presence of S653D SNP and another 145 F2 individuals from natural red rice × CL121 hybridizations were screened for the presence of the G654E SNP. The PCR-based assays produced during this study are simple, rapid, inexpensive, reproducible and require only standard PCR and electrophoretic instruments that can be applied toward outcrossing evaluation and effective weed management strategies for the Clearfield crop system. [ABSTRACT FROM AUTHOR]

The Latvian and the Swedish sweet cherry ( Prunus avium L.) genetic resources collections comprise valuable material for breeding. The collections represent local Latvian and Scandinavian genetic resources: semi-wild samples, landraces, and cultivars developed in local breeding programmes, as well as diverse germplasm from the northern temperate zone. The objective of this investigation was to determine which S 1 –S 6 alleles are most important in the sweet cherry genetic resources collections and to compare the identified allelic and genotypic frequencies in material of different origin. Accessions in the two collections were screened for the presence of the self-incompatibility ( S) S 1 to S 6 alleles, using PCR based typing. Significant differences ( P < 0.05) between screened collections were found in frequencies of S 4 and S 5 alleles. Analysis of allele combinations identified the high occurrence of selections with the S-genotype S 3 S 6 in both collections. Compared to the S-allele frequencies published for over 250 sweet cherry cultivars from Western and Southern Europe, the Latvian and Swedish germplasm appeared to have a high frequency of the S 6 allele in both collections, and a relatively high frequency of the S 5 allele in Latvian germplasm. This study represents the first comprehensive S-allele screening for the sweet cherry genetic resources collections in Latvia and Sweden. Both sweet cherry collections contain high proportion of accessions adapted to north central European growing conditions, not typical for the majority of the documented sweet cherry genetic resources, which explains differences in certain S-allele occurrence. [ABSTRACT FROM AUTHOR]

Objective Particularly for therapy-experienced patients, resistance assessment by genotypic or phenotypic methods produces discordances. This study seeks proof that differences may arise from the fact that genotyping produces a single summary sequence whereas replicative phenotyping (rPhenotyping) functionally detects and assigns resistances in mixed HIV populations. Methods For validation, defined mixes of wild-type and M184V mutant were analysed by rPhenotyping or standard genotyping. Allele-specific and quantitative polymerase chain reaction (PCR) set detection and quantification limits for minor virus populations in vitro and in authentic clinical samples showing geno-/pheno-discrepant lamivudine resistance. Results Allele-specific and real-time PCR methods detected down to 0.3% of mutant M184V. The functional assessment was sensitive enough to reveal <1% of mutant M184V in mixed samples. Also in discordant samples from the diagnostic routine, in which rPhenotyping had identified drug resistance, real-time PCR confirmed minute amounts of mutant M184V. Conclusion By utilizing the replication dynamics of HIV under drug pressure, a rPhenotyping format potently reveals relevant therapy-resistant minority species, even of HIV known to possess reduced replicative fitness. With its rapid turnaround of 8 days and its high sensitivity, our rPhenotyping system may be a valuable diagnostic tool for detecting the early emergence of therapy-threatening HIV minorities or the persistence of residual resistant virus. [ABSTRACT FROM AUTHOR]

Abstract: Allele-specific PCR (ASPCR) is a highly sensitive, and reproducible method for the study of minor HIV-1 variants harboring resistance mutations and is significantly less labor-intensive and time-consuming than other techniques used for similar purposes. Furthermore, ASPCR has multiple applications in HIV research: it provides earlier and more sensitive detection of evolving resistance mutations, a more accurate assessment of transmitted drug-resistant mutants and a better evaluation of resistance selection after post-exposure or mother-to-child-transmission prophylaxis programs. This article outlines the principles of ASPCR and illustrates technical challenges in the design and application of ASPCR protocols by describing ASPCR assays developed for detecting resistance mutations in the protease (PR)- and reverse transcriptase (RT)-coding regions of pol and env. The assays achieved sensitivities of <1% for the D30N mutation in HIV-1 PR, M184V and I mutations in RT, and V38A in gp41. This method can be easily adapted to the quantitative detection of other mutations in HIV-1 or other viruses by introducing minor modifications to the methods described. In addition, ASPCR can be used to assess the dynamics of mutant populations in the viral quasispecies in response to changing selection pressures, allowing inferences on viral fitness in vivo through mathematical modeling. [Copyright &y& Elsevier]

The so-called Rosa (= pink) tomatoes, which are typically grown in the Southern Italian area, are characterized by the pink colour of the fruit, due to the gene y, colourless fruit skin. In a preliminary survey, it was found that among these Rosa tomatoes there were some ‘Rosa di Sorrento’ local landraces showing resistance to Verticillium wilt (race 1). In tomato, resistance to race 1 of V. dahliae and V. albo-atrum is conferred by two strictly associated genes, Ve1 and Ve2, which independently confer resistance to the same pathogen. The development of two new markers for Ve1 and Ve2, based respectively on selective allele-specific PCR amplification and on a PCR amplification followed by enzymatic restriction, is reported. These two markers allow the identification of both allelic forms at the Ve loci and they are of potential interest for use in marker-assisted selection. Furthermore, ‘Rosa di Sorrento’-resistant lines have the same resistance alleles as those found in the Ve-resistant cultivars. [ABSTRACT FROM AUTHOR]

Objective To assess the application of allele-specific PCR (AS-PCR) as a fast, cheap and reliable method for detecting mutant TbAT1 associated with melarsoprol relapse in Trypanosoma brucei gambiense isolates from northwest Uganda. Methods A total of 105 trypanosome isolates were analysed using SfaN1 restriction fragment length polymorphism (RFLP) and AS-PCR, the former used as the gold standard. Sensitivity, specificity, positive and negative predictive values of AS-PCR as well as agreement between the tests were determined. Results Eleven trypanosome isolates had mutant TbAT1 while 94 exhibited the wild-type TbAT1 genes. There was a highly significant agreement between SfaN1 RFLP and AS-PCR with kappa and intra-class correlation values of 1.0. The sensitivity and specificity of AS-PCR were both 100%, while the positive and negative predictive values were found to be equal to 1.0. Cost and time analyses were performed and AS-PCR was 4.3 times cheaper than SfaN1 RFLP, in addition to the less time required for its execution. Conclusion AS-PCR should be the test of choice for screening for mutant TbAT1 in the ever-increasing numbers of field trypanosome isolates. [ABSTRACT FROM AUTHOR]

Abstract: We report the use of capillary gel electrophoresis (CGE) based on a rfbS allele-specific polymerase chain reaction (PCR) for the analysis and simultaneous detection of Salmonella pullorum and Salmonella gallinarum, which are the major bacterial pathogens in poultry. rfbS allele-specific PCR was used to concurrently amplify two specific 147- and 187-bp DNA fragments for the simultaneous detection of S. pullorum and S. gallinarum at an annealing temperature of 54±1°C and an MgCl2 concentration of 2.8–5.6mM. Under an electric field of 333.3V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) (M r 600000), the amplified PCR products were analyzed within 6min by CGE separation. This CGE assay could be translated to microchip format using programmed field strength gradients (PFSG). In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30s, without decreasing the resolution efficiency. rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique might be a new tool for the simultaneous detection of both S. pullorum and S. gallinarum, due to its ultra-speed and high efficiency. [Copyright &y& Elsevier]

Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047–0.05 for CytB, 0.084–0.166 for ITS1 and 0.03–0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique. [ABSTRACT FROM AUTHOR]

Abstract: To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5′-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5′-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure. [Copyright &y& Elsevier]

Abstract: Background: The genotyping for the study of the SNPs in different complex diseases require a great number of patients. In this sense, the determination of allele frequencies and genotypes requires a rapid and economical procedure. Methods: The genotype has been carried out by allele-specific PCR in single tube with the discrimination of the products of PCR by its T m. For this purpose a GC tail was added to 5′ extreme of the specific primer. The allele frequencies were also calculated by DNA pooling and QRT-PCR using allele-specific primers. Results: The use of the genotyping in single tube through allele-specific PCR and melting curves has led us to the accurate genotype of three polymorphisms of vdr (cdx-2), osteoprotegerin (A-163G) and ppar-γ (C-681G) genes in 225 postmenopausal women to be associated to osteoporosis. Only the cdx-2 polymorphism was associated with a reduced bone mineral density (BMD). These data were similar to those obtained when the allele frequencies were calculated using QRT-PCR in DNA pools. Conclusions: Individual genotyping with allele-specific PCR in single tube and melting curve analysis is a fast, trustworthy and economic method to study any SNP. We propose the following approach to determine the possible association of SNPs with complex and multifactorial diseases like osteoporosis, in which hundreds of individuals should be analyzed: construct control and problem groups, make DNA pools, and calculate pooled allelic frequencies. Genotyping each individual further permits to determine the genotypic distribution when differences in allelic frequencies are observed, thus allowing more complex statistical analyses (including other variables like age, weight, etc.). [Copyright &y& Elsevier]

During the investigation of a CYP2C8*3 genetic polymorphism in a South American population, we obtained a discrepant result using two different polymerase chain reaction (PCR) protocols. A single nucleotide polymorphism (SNP) (IVS3+43 G>C) was identified in the intron 3 region, which was used as a primer-annealing site of one of the two PCR protocols. A genotyping method was developed to enable discrimination of the CYP2C8*1A, CYP2C8*3 (416 G>A), and CYP2C8*3 (416G>A; IVS3+43 G>C) alleles. In a screen of a South American population, we found that individuals carrying the CYP2C8*3 (416 G>A) polymorphism also carried the CYP2C8*3 (416G>A; IVS3+43 G>C). However, we did not find any carriers of CYP2C8*3 (416G>A; IVS3+43 G>C) in a Japanese population. [ABSTRACT FROM AUTHOR]