Nonradioactive Method for Quantitation of PCR Products Without Hybridization with a Specific Probe

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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Enterovirus D-68 Molecular Virology, Epidemiology, and Treatment: an update and way forward

Infectious Disorders - Drug Targets ◽

10.2174/1871526520666200715101230 ◽

2020 ◽

Vol 20 ◽

Author(s):

Mahmoud Ahmed Ebada ◽

Notila Fayed ◽

Souad Alkanj ◽

Ahmed Wadaa Allah

Keyword(s):

Current Knowledge ◽

Rna Virus ◽

Neurological Diseases ◽

Cross Reactivity ◽

Serological Tests ◽

Molecular Virology ◽

Acute Flaccid Myelitis ◽

The Family ◽

Pcr Products ◽

Enterovirus D68

: Enterovirus D68 (EV-D68) is a single-stranded positive-sense RNA virus, and it is one of the family Picornaviridae. Except for EV-D68, the family Picornaviridae has been illustrated in literature. EV-D68 was first discovered and isolated in California, USA, in 1962. EV-D68 has resulted in respiratory disorders’ outbreaks among children worldwide, and it has been detected in cases of various neurological diseases such as acute flaccid myelitis (AFM). A recent study documented a higher number of EV-D68 cases associated with AFM in Europe in 2016 compared to the 2014 outbreak. EV-D68 is mainly diagnosed by quantitative PCR, and there is an affirmative strategy for EV-D68 detection by using pan-EV PCR on the untranslated region and/or the VP1 or VP2, followed by sequencing of the PCR products. Serological tests are limited due to cross-reactivity of the antigens between the different serotypes. Many antiviral drugs for EV-D68 have been evaluated, and showed promising results. In our review, we discuss the current knowledge about EV-D68 and its role in the development of AFM.

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Monoclonal anti-light chain idiotype as a tumor-specific probe for human neoplastic B lymphocytes

Blood ◽

10.1182/blood.v69.3.919.919 ◽

1987 ◽

Vol 69 (3) ◽

pp. 919-923 ◽

Cited By ~ 4

Author(s):

M Wrightham ◽

AL Tutt ◽

MJ Glennie ◽

TJ Hamblin ◽

GT Stevenson ◽

...

Keyword(s):

B Cell ◽

Tumor Cells ◽

B Lymphocytes ◽

Light Chain ◽

Normal Serum ◽

Lymphocytic Leukemia ◽

Light Chains ◽

Specific Probe ◽

Tonsillar Cells ◽

Binding Curves

Abstract Tumor cells from patients with B cell neoplasms often secrete small amounts of free monoclonal light chains that can be found in the urine. Such tumor-derived light chains of the lambda type from a patient with typical chronic lymphocytic leukemia have been used to raise mouse monoclonal antibodies (MoAbs). A hybridoma-secreting antibody that recognized the idiotypic lambda chain but not normal lambda chains by a preliminary screen but which also reacted with idiotypic IgM from the patient's tumor cells was selected. This MoAb in fact recognized 1 in 20 X 10(3) molecules of pooled normal lambda chains, thus establishing its specificity for a private idiotypic determinant. It failed to give a detectable reaction with normal IgM, normal serum, or a panel of IgM paraproteins. The antibody bound to the patient's neoplastic B cells but not to normal tonsillar cells. The site of binding of the antibody to idiotypic IgM is clearly separate from that of another MoAb specific for idiotypic determinants on heavy plus light chains, since the two showed additive binding curves. The determinant also appeared to be less available in dimeric lambda chains than in monomeric lambda chains or in idiotypic IgM. Antibodies to idiotypic determinants on light chains show some technical advantages and should be useful for monitoring and possibly treating B cell tumors, either alone or together with the more conventional anti-idiotypic antibodies that usually recognize the heavy and light chain combination.

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Parathyroid Ca2+-conducting currents are modulated by muscarinic receptor agonists and antagonists

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1997.273.5.e880 ◽

1997 ◽

Vol 273 (5) ◽

pp. E880-E890 ◽

Cited By ~ 1

Author(s):

Wenhan Chang ◽

Tsui-Hua Chen ◽

Stacy A. Pratt ◽

Benedict Yen ◽

Michael Fu ◽

...

Keyword(s):

Muscarinic Receptors ◽

Inositol Phosphate ◽

Receptor Protein ◽

Dependent Manner ◽

Rt Pcr ◽

Pipette Solution ◽

Pcr Products ◽

Inositol Phosphate Accumulation ◽

Receptor Cdna ◽

Dose Dependent

Parathyroid cells express Ca2+-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration ≈10−8 M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution. (+)-Muscarine enhanced the adenosine 3′,5′-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed >90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.

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Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Gut Pathogens ◽

10.1186/s13099-021-00431-7 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Sohyun Lee ◽

Nanjoo Park ◽

Sujung Yun ◽

Eunseon Hur ◽

Jiwon Song ◽

...

Keyword(s):

South Korea ◽

Pcr Amplification ◽

Public Health Problem ◽

Test Method ◽

Quinolone Resistance ◽

Human Salmonellosis ◽

Pcr Products ◽

Antimicrobial Susceptibility Tests ◽

Susceptibility Tests ◽

Sequence Types

AbstractNon-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.

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173 The role of an intronic single nucleotide polymorphism within the dopamine receptor type-2 gene on pituitary prolactin protein expression in bulls

Journal of Animal Science ◽

10.1093/jas/skz397.085 ◽

2020 ◽

Vol 98 (Supplement_2) ◽

pp. 37-37

Author(s):

Andrea N DeCarlo ◽

Keelee J McCarty ◽

Sarah K Richey ◽

Nathan Long ◽

Scott Pratt

Keyword(s):

Protein Expression ◽

Genotype Distribution ◽

Nucleotide Polymorphisms ◽

Genotypic Effect ◽

Single Nucleotide ◽

Detection Range ◽

Cull Cows ◽

Densitometry Analysis ◽

Pcr Products

Abstract Detrimental effects to male reproductive physiology have been observed due to changes in prolactin (PRL) serum concentration. Regulation of PRL by dopamine binding to the dopamine type-2 receptor (DRD2) is well defined and associations between male physiology and single nucleotide polymorphisms (SNPs) within the DRD2 gene have been observed. The objective of the study was to evaluate association of a DRD2 SNP to PRL protein expression in bulls. Testis and epididymis were collected from bulls grazing a forage containing or lacking a dopamine agonist at the end of a 126 d study (n = 14). Bovine pituitaries (n = 587) were collected randomly over 3 mo from a local abattoir which processes cull cows and bulls. Sex of pituitaries was verified (n = 259 males) by duplex PCR for amplification of SRY and b-actin followed by Southern blotting of PCR products for selection of male. Prolactin protein expression was assessed in testis, epididymis, and pituitary by western blotting. Expression of PRL protein was below detection range in reproductive tissues but was present in pituitary, therefore experiments continued in pituitary. Restriction fragment length polymorphism genotyping was performed on pituitaries by amplification of the DRD2 SNP region followed by digestion with a Tfil enzyme. Digested of products produced 3,2, or 1 band (AG, AA, GG, respectively). A subset of male pituitaries was blotted by slot blot manifold and PRL protein expression assessed by immunodetection and densitometry analysis normalized to GAPDH expression. Pituitary genotype distribution was 17.4% AA (n = 16), 63% AG (n = 58), and 19.6% GG (n = 18). Prolactin protein expression in the pituitary was similar across genotype (P = 0.23). These findings indicate that the DRD2 SNP has no genotypic effect on PRL protein expression in bovine pituitary.

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