scholarly journals PIRA-PCR for Detection of Fusarium fujikuroi Genotypes with Carbendazim-Resistance Alleles

Plant Disease ◽  
2015 ◽  
Vol 99 (9) ◽  
pp. 1241-1246 ◽  
Author(s):  
Zhen Zhang ◽  
Zihao Chen ◽  
Yiping Hou ◽  
Yabing Duan ◽  
Jianxin Wang ◽  
...  
Keyword(s):  
Restriction Analysis ◽  
Point Mutations ◽  
Enzyme Digestion ◽  
Gibberella Fujikuroi ◽  
Fusarium Fujikuroi ◽  
Chain Reaction ◽  
Bakanae Disease ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Enzyme Recognition

Carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide, has been used to control rice bakanae disease, caused by Fusarium fujikuroi (teleomorph: Gibberella fujikuroi), for decades in China. Previous research revealed that point mutations (E198V, GAG to GTG at codon 198, and F200Y, TTC to TAC at codon 200) of the β2-tubulin gene conferred resistance of F. fujikuroi to MBC. In this study, primer-introduced restriction analysis polymerase chain reaction (PIRA-PCR) was developed to determine genotypes with resistance of F. fujikuroi to MBC. A PCR template of each strain was created by an outer primer pair. Fragments with 177 bp (for mutation at codon 235) and 146 bp (for E198V) were amplified by nested PCR, with two inner primer pairs designed and synthesized according to the nucleotide sequence of β2-tubulin for further enzyme digestion validation, respectively. AccII and PmaCI restriction enzyme recognition sites were introduced artificially by inner primers to differentiate MBC-sensitive and -resistant strains, respectively. The sensitivity of each strain to MBC was indirectly determined by analyzing electrophoresis patterns of the resulting amplified fragments after simultaneous digestion by both AccII and PmaCI. PIRA-PCR produced the same result as conventional methods in 6% of the time. PIRA-PCR is a sensitive and effective method for genotyping resistance alleles of F. fujikuroi strains to MBC.

Detection of the Glanzmann's Thrombasthenia Mutations in Arab and Iraqi-Jewish Patients by Polymerase Chain Reaction and Restriction Analysis of Blood or Urine Samples

1991 ◽  
Vol 66 (04) ◽  
pp. 500-504 ◽  
Author(s):  
H Peretz ◽  
U Seligsohn ◽  
E Zwang ◽  
B S Coller ◽  
P J Newman
Keyword(s):  
Polymerase Chain Reaction ◽  
Base Pair ◽  
Restriction Analysis ◽  
Urine Samples ◽  
Chain Reaction ◽  
Base Pairs ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Base Pair Deletion ◽  
Polymerase Chain

SummarySevere Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews – an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs – a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

Norwalk-like Virus Sequences Detected by Reverse Transcription-Polymerase Chain Reaction in Mineral Waters Imported into or Bottled in Switzerland

2000 ◽  
Vol 63 (11) ◽  
pp. 1576-1582 ◽  
Author(s):  
CHRISTIAN BEURET ◽  
DOROTHE KOHLER ◽  
THOMAS LÜTHI
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Carbonic Acid ◽  
Point Mutations ◽  
Chemical Properties ◽  
The United States ◽  
Mineral Waters ◽  
Nucleotide Sequence Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

Norwalk-like viruses (NLVs) is a genus belonging to the Caliciviridae. NLVs are transmitted by the fecal-oral and the aerosol route and are the most common cause of outbreaks of nonbacterial gastroenteritis. NLVs are responsible for an estimated 67% of all illnesses caused by known foodborne pathogens and for 96% of nonbacterial gastroenteritis in the United States. Many outbreaks could be associated with the consumption of primarily or secondarily contaminated foods. To our knowledge, no epidemic arising from contaminated mineral water has been reported. We investigated the presence of NLV sequences in 63 mineral waters of 29 different brands that were imported into or bottled in Switzerland. NLV sequences were detected in 21 mineral waters by reverse transcription-seminested polymerase chain reaction. Specimens of two NLV genogroups (gg), gg I and gg II, were randomly present in the contaminated samples. The presence of NLV sequences could not be correlated either with bottle characteristics or with chemical properties like mineralization, pH, or the presence of carbonic acid. Nucleotide sequence analysis of 12 NLV-positive samples revealed several point mutations. All isolated NLV gg I strains have a similarity of 70 to 87% with the common Desert Shield virus (UO4469), and all isolated NLV gg II strains have a similarity of 89 to 93% with the Camberwell virus (U46500). Possible reasons for the presence of NLV sequences in mineral waters are discussed.

scholarly journals Precision of genotyping of Epstein-Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C, and EBER): predominance of type A virus associated with Hodgkin's disease [published erratum appears in Blood 1993 Oct 1;82(7):2268]

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3372-3381 ◽  
Author(s):  
JC Lin ◽  
SC Lin ◽  
BK De ◽  
WC Chan ◽  
BL Evatt ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Point Mutations ◽  
Type A ◽  
Type B ◽  
Chain Reaction ◽  
Barr Virus ◽  
Polymerase Chain ◽  
Epstein Barr

Abstract To precisely determine the genotype of Epstein-Barr virus (EBV) in Hodgkin's disease (HD), we simultaneously analyzed three divergent gene loci (EBNA-2, EBNA-3C, and EBER) that distinguish type A and B viruses. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of the polymerase chain reaction (PCR)-amplified products or the mobility shifts in single-strand conformation polymorphism analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. We analyzed 15 EBV-infected cell lines and found a good correlation between EBNA-2 and EBNA-3C typing results. In contrast, approximately 33% of the cell lines analyzed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B, and 31% with dual viral sequences. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis, and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.

scholarly journals Detection of occult follicular lymphoma by specific DNA amplification

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1822-1825 ◽  
Author(s):  
M Stetlet-Stevenson ◽  
M Raffeld ◽  
P Cohen ◽  
J Cossman
Keyword(s):  
Follicular Lymphoma ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Direct Application ◽  
Chain Reaction ◽  
Heat Stable ◽  
Valuable Adjunct ◽  
Polymerase Chain

Abstract To detect occult lymphoma, the polymerase chain reaction (PCR) technique was used to amplify joined bcl-2/JH DNA sequences at the juncture of the t(14:18) translocation in follicular lymphoma. Using the heat-stable DNA polymerase Taq and automated cycling of the reaction, we were able to detect as few as one to two copies of bcl- 2/JH. Under these conditions, PCR proved to be at least 10,000-fold more sensitive than either conventional flow cytometry or Southern blot restriction analysis. In addition, genomic DNA sequences of four lymphomas confirmed that the size of the amplified segment serves as a tumor marker. Direct application of PCR to patient staging revealed occult malignant lymphoma in tissue otherwise considered uninvolved by standard criteria. We conclude that the striking enhancement in diagnostic sensitivity attained by DNA amplification can serve as a valuable adjunct to the staging and clinical monitoring of follicular lymphoma.

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