Universal fluorescent labeling of PCR products for DHPLC analysis: reducing cost and increasing sample throughput

ABSTRACT Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.

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Automated High-Throughput Purification of PCR Products Using Wizard®MagneSil™ Paramagnetic Particles

JALA Journal of the Association for Laboratory Automation ◽

10.1016/s1535-5535(04)00232-1 ◽

2002 ◽

Vol 7 (6) ◽

pp. 120-124

Author(s):

P Otto

Keyword(s):

High Throughput ◽

Pcr Products

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Abnormal Processing of the Glycoprotein IIb Transcript due to a Nonsense Mutation in Exon 17 Associated with Glanzmann’s Thrombasthenia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653864 ◽

1995 ◽

Vol 73 (05) ◽

pp. 756-762 ◽

Cited By ~ 25

Author(s):

Yoshiaki Tomiyama ◽

Hirokazu Kashiwagi ◽

Satoru Kosugi ◽

Masamichi Shiraga ◽

Yoshio Kanayama ◽

...

Keyword(s):

Dna Analysis ◽

Molecular Genetic ◽

Genetic Defect ◽

Nucleotide Sequence Analysis ◽

Type I ◽

Normal Amount ◽

Immunoblot Assay ◽

Glanzmann’S Thrombasthenia ◽

Glanzmann's Thrombasthenia ◽

Pcr Products

SummaryWe analyzed the molecular genetic defect responsible for type I Glanzmann’s thrombasthenia in a Japanese patient. In an immunoblot assay using polyclonal anti-GPIIb-IIIa antibodies, some GPIIIa (15% of normal amount) could be detected in the patient’s platelets, whereas GPIIb could not (<2% of normal amount). Nucleotide sequence analysis of platelet GPIIb mRNA-derived polymerase chain reaction (PCR) products revealed that patient’s GPIIb cDNA had a 75-bp deletion in the 3’ boundary of exon 17 resulting in an in-frame deletion of 25 amino acids. DNA analysis and family study revealed that the patient was a compound heterozygote of two GPIIb gene defects. One allele derived from her father was not expressed in platelets, and the other allele derived from her mother had a 9644C → T mutation which was located at the position -3 of the splice donor junction of exon 17 and resulted in a termination codon (TGA). Moreover, quantitative analysis demonstrated that the amount of the abnormal GPIIb transcript in the patient’s platelets was markedly reduced. Thus, the C → T mutation resulting in the abnormal splicing of GPIIb transcript and the reduction in its amount is responsible for Glanzmann’s thrombasthenia.

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MOLECULAR STUDY OF HUMAN HEAD LICE, PEDICULUS HUMANUS CAPITIS COMPARISON WITH GOAT SUCKING LICE, LINOGNATHUS STENOPSIS (BURMEISTER) STRAINS

Anbar Journal of Agricultural Sciences ◽

10.32649/ajas/2020/12 ◽

2020 ◽

pp. 132-139

Keyword(s):

Genetic Difference ◽

Human Head ◽

Molecular Study ◽

Head Lice ◽

Pediculus Humanus Capitis ◽

School Students ◽

Pediculus Humanus ◽

Sucking Lice ◽

Pcr Products ◽

Amplicon Size

In this study, only (122) out of (915) primary school students were shown to be infected with head lice Pediculus. humanus capitis. The number and percentage of infected males were 46 (11.3%), while the number and percentage of infected females were 76 (14.9%). The results in our study also showed that the number and percentage of goats infected with goat sucking lice, Linognathus stenopsis was 70 (21.7%) of the total 322 animals, with the highest number and percentage among female goats 44 (62.9%) compared to the male goats 26 (37.1%). The study demonstrated that the rate of genetic difference between the studied samples was 89% and the similarity rate was 11%. Detection of OP-K01 gene pieces by PCR products showed that the amplicon size was 520 bp for P. humanus capitis isolated from humans, while the detection of OP-E20 and OP-M05 gene pieces with PCR product showed the lowest amplicon size 230 bp for Linognathus stenosis isolated from goats.

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Linkage between H. pylori Infection and TNF-α polymorphism in The Pregnant Women

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v9ispl1.1298 ◽

2018 ◽

Vol 9 (SPL1) ◽

Author(s):

Ghaidaa Raheem Lateef ◽

Azhar Omaran Al-Thahab

Keyword(s):

Pregnant Women ◽

Serum Antibody ◽

Rapid Test ◽

Negative Group ◽

Positive Group ◽

Gynecology And Obstetrics ◽

Tnf Α ◽

Significant Difference ◽

Pcr Products ◽

H Pylori

A study was performed on 100 pregnant women in the outpatient department of gynecology and obstetrics of Maternity and Children Hospital in Al-Diwaniya City during the period between (March to September 2016). One hundred blood samples (50 for patients and 50 for control) were collected under the supervision of the treating gynecologist. The detection of Helicobacter. pylori was done by the use of the serum antibody Rapid test. The results showed that 50 (100%) were positive and 50 (100%) were negative for H. pylori in above method.All blood of patients and control samples were used for the extraction of genomic DNA,where the 107 bp PCR product size. Genotyping of the TNF-α-308 SNP (G/A)was performed by restriction fragment length polymorphism PCR (RFLP-PCR). PCR products were digested with restr NcoI iction enzyme. Individuals with the TNF-α-308(GG) homozygote produced digested DNA bands at 80,and 20 bp bp. A heterozygous genotype ofTNF-α-308 (GA)produced 107 bp,80 bp,and 20 bp bands. Individuals with the TNF-α-308 (AA) homozygote genotype had no amplicon digested and generated only one band of 107 bp. There was a significant difference in the frequency of the TNF-α-308(GG)genotype between H. pylori positive group and H. pylori negative group(72%,78% respectively). Also for GA genotype,there was a significant difference between H. pylori positive group and H. pylori negative group(24%,18% respectively). Concerning the frequency of the TNF-α-308 (AA)genotype between H. pylori positive group and H. pylori negative group,there was no significant difference between the two groups.

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MICROSCOPIC AND MOLECULAR DIAGNOSIS OF Ascaridia spp. IN DOMESTIC PIGEONS(Columba livia domestica) IN BAGHDAD CITY,IRAQ

IRAQI JOURNAL OF AGRICULTURAL SCIENCES ◽

10.36103/ijas.v51i4.1101 ◽

2020 ◽

Vol 51 (4) ◽

pp. 1220-1225

Author(s):

Faraj & Al- Amery

Keyword(s):

Molecular Diagnosis ◽

Columba Livia ◽

Ascaridia Galli ◽

Molecular Assay ◽

Infection Rates ◽

Conventional Pcr ◽

Significant Difference ◽

Pcr Products ◽

Males And Females ◽

Domestic Pigeons

Ascaridiosis is a very important parasitic disease of birds, it is caused by Ascaridia. This study was conducted to identify the Ascaridia species by microscopic and molecular assay in Baghdad city. One hundred and sixty fecal samples were collected from domestic pigeons during the period from 1/1/ 2019 to 31/3/ 2019. Results showed that the rate of infection for Ascaridia spp. 15.62% by microscopic examination. Significant difference was observed in infection rates between males and females pigeons. Fifty samples randomly selected and subjected to molecular diagnosis of Ascaridia spp.. Molecular examination results, the total infection rate showed 16%(8/50). The eight positive PCR products were sequenced and deposited in Gene bank data base, phylogenic analysis demonstrated that 4 sequences belongs to Ascaridia galli ( MK918635.1, MK918636.1, MK918847.1, MK919081.1), while 2 (MK919199.1, MK919200.1) belong to Ascaridia nymphii and 2 (MK919207.1, MK919264.1) belong to Ascaridia numidae. It is the first study in Iraq to diagnosis of Ascaridia nymphii and Ascaridia numidae in domesticed pigeons by using conventional PCR.

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