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1.
Anubha Paliwal Nivedita Bir Sowmya Raghuraman T. L. P. Reddy P. Usha Sarma 《Indian journal of clinical biochemistry : IJCB》1999,14(2):129-134
Oligonucleotide primers were synthesised based on the gene sequence of an 18 kDa allergen/antigen ofA. fumigatus isolated from a pathogenic strain. Polymerase chain reaction (PCR) was carried out using the forward and reverse primers
and genomic DNA ofA. fumigatus, A. flavus andA. niger as template. This resulted in a PCR product of 480 bp with onlyA. fumigatus. The absence of PCR product inA. flavus andA. niger with the primers of Asp fl facilitated use of these primers for detection ofA. fumigatus in clinical specimens of patients. The results were compared with microscopy, culture and serology. Application of PCR test
to clinical samples of aspergillosis patients is discussed. 相似文献
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Ritu Singh Vijjaya Madhulika Kabra 《Indian journal of clinical biochemistry : IJCB》2006,21(1):147-151
Duchenne muscular dystrophy (DMD) is the most common X-linked disorder in children affecting 1 in 3500 males. Since, as of
now, we have no treatment for DMD, carrier detection and prenatal diagnosis is the most important preventive strategy. Multiplex
PCR helps in rapid detection of hot spot exonal deletions (positive in 65% of cases) as many exons can be identified in a
single run. 10 children with characterstic clinical features of DMD and chorionic villus samples of 10 antenatal patients
with positive family history were studied. We identified a deletion mutation in exon 49 of the dystrophin gene in a 4 yr old
boy referred with signs and symptoms suggestive of DMD using primers for exons 45, 48, 49, 43, 44, 19, 3, 8, 13 and muscle
promoter, subjected to multiplex polymerase chain reaction (PCR) and agarose/Nu-Sieve gel electrophoresis. These genetic methods
aid in prenatal diagnosis of DMD as well as confirmation of diagnosis in children with signs and symptoms suggestive of the
disease.
Work done as WHO fellow in Deptt. of Genetics, All India Institute of Medical Sciences, New Delhi. 相似文献
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为研究斜纹夜蛾核型多角体病毒(Spodoptera litura muhicapsid nucleopolyhedrovirus.SpltMNPV)侵染规律,根据SphMNPV中国株(G2)ORF135基因序列设计引物,经PCR扩增,从SpltMNPV日本株(B)基因组中克隆了pif-2基因。核苷酸序列分析表明,该基因是甜菜夜蛾核型多角体病毒(Spodoptera exigua nucleopolyhedrovirus,SeMNPV)pif-2的同源物,读码框含1278bp,编码425个氨基酸的蛋白质,推定分子量为48.6kD。与其他杆状病毒的同源物比对显示.SpltMNPV-JP-B PIF-2蛋白中14个Cys残基高度保守。联配分析表明,SphMNPV-JP-B pif-2的核苷酸序列与其他13种核型多角体病毒的同源性有较大差异。将pif-2克隆至原核表达载体pET32a(+),经IPTG诱导后在大肠杆菌BL21中获得了融合表达,SDS-PAGE分析表明在约69kD处有特异性表达条带,经Western blot验证该蛋白即为融合表达的目的蛋白。 相似文献
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Manjusha Dixit Anvesha Srivastava Gourdas Choudhuri Balraj Mittal 《Indian journal of clinical biochemistry : IJCB》2008,23(2):123-129
Background: Imbalance in cholesterol homeostasis may lead to gallstone disease. Apolipoprotein B is sole component of low-density
lipoprotein and plays an important role in cholesterol metabolism. The present study was carried out to explore the association
of APOB 3′ VNTR, exon 26 XbaI and signal peptide insertion/ deletion polymorphisms with gallstone disease. 214 ultrasonographically proven gallstone patients
and 322 healthy, age and sex matched controls were taken for the study. Genotyping was done using PCR followed by polyacrylamide
gel electrophoresis for VNTR and insertion/ deletion analysis. For APOB XbaI polymorphism PCR product was digested with XbaI restriction enzyme, followed by agarose gel electrophoresis. All statistical analyses were done using SPSS v11.5. Higher
repeat alleles of APOB 3′ VNTR polymorphism were more frequent in gallstone patients than in controls. Alleles with more than 57 repeats were present
only in patient group. Long (L) alleles with repeat higher than 49, were significantly higher (P=0.000; OR=3.705, 95% CI 2.577–5.326) and medium (M) alleles were lower (P=0.000; OR=0.406, 95% CI 0.304–0.542) in patients than in controls. To nullify the effect of gender, data was further stratified
into male and female population. APOB 3′ VNTR, L alleles were imposing risk and M alleles were protective in both male and female population. APOB
XbaI and insertion/deletion polymorphisms were not found to be associated with the gallstone disease. Longer alleles of APOB 3′ VNTR occur more frequently in gallstone patients, and may be an important risk factor for the development of gallstone
disease. APOB XbaI and signal peptide insertion/deletion polymorphisms may not be contributing to the risk for gallstone disease. 相似文献
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Poonam Gautam Taruna Madan W. N. Gade P. Usha Sarma 《Indian journal of clinical biochemistry : IJCB》2006,21(2):12-19
Allergenic/antigenic proteins are known to induce Type I and Type III hypersensitivity reactions leading to allergic bronchopulmonary
aspergillosis (ABPA) in immunocompetent host. The common structural features or intrinsic properties of the allergens/antigens
leading to allergenicity in a host are not well understood. In the current report, comparative analysis of proteins on two
dimensional gel electrophoresis (2D-3) and specific IgE immunoblots ofA. fumigatus secretory proteins (1st, 2nd and 3rd week culture filtrate proteins) was carried out. We observed a total of 159 proteins in 1st, 2nd and 3rd week culture filtrates ofA. fumigatus. Specific IgE immunoreactivity was observed in 75 proteins with different intensity. Third week culture filtrate showed maximum
number of proteins, 142, and specific IgE immunoreactive proteins, 65. MALDI-TOF analysis resulted in putative identification
of two allergens as hypothetical protein YBL057c fromSaccharomyces cereviseae and unnamed protein product fromDebaryomyces hansenii (similar to IPF14568 ofCandida albicans). Identification of a repertoire of specific IgE immunoreactive proteins will facilitate the studies on structure-function
relationship of these proteins relevant for diagnosis and pathogenesis. 相似文献
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Y. P. Bhandary K. N. Krithika Sandeep Kulkarni M. V. R. Reddy B. C. Harinath 《Indian journal of clinical biochemistry : IJCB》2006,21(1):1-7
Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key
enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase
(G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high
in mf lysate compared to their activity in lysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel
to their activity inlysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis (PAGE)
using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes (MDH, ME & G6PDH) in mf lysate,
while only one form of each enzyme was present in L3 larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval
lysates ofB. malayi. While both mf and L3 larval lysates showed optimal protease activity at alkaline pH of 9.0, the mf lysate showed increased activity also at pH
3.0. The infective larval lysate was markedly inhibited by Tosylamide-L-Phenylalanine chloromethyl ketone (TPCK), a thiol
protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease
inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),
using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the
L3 larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size. 相似文献
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Ipsita Choudhury R. Jothimalar Arun Kumar Patra 《Indian journal of clinical biochemistry : IJCB》2012,27(3):265-269
Hypertension, a well known risk factor for various cardiovascular, peripheral vascular and renal events is an important public health challenge. Renin angiotensin system (RAS) being the most vital pathogenic mechanism of hypertension is mediated by a key component; the angiotensin converting enzyme (ACE). The present study was aimed to know the relationship of ACE gene polymorphism and the possible risk of development of hypertension in south Indian population. The study included 101 clinically diagnosed hypertensive patients without any associated disease condition and 81 age and sex matched apparently healthy controls. Genotyping was performed using a polymerase chain reaction, (PCR) amplification of the intron 16 fragment harboring the 287 bp Alu repeat sequence. Three possible genotypes D/D, I/I homozygous and I/D heterozygous were analyzed where the D/D genotypes corresponds to higher ACE levels (D-Deletion, I-Insertion). The PCR products were separated on 2 % agarose gel. Statistical analysis was performed using SPSS.15 software program. We found a significance in frequency of D/D genotype in the hypertensive patients compared to the control group (p = 0.0005, odd’s ratio = 4.157). This suggested that ACE (D/D) genotypes are more prone for the development of hypertension. This is relatively a pilot study; but nevertheless may assist in identifying the pathophysiological cause of hypertension. 相似文献
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Linda Desbois Adrien Padirac Shohei Kaneda Anthony J. Genot Yannick Rondelez Didier Hober Dominique Collard Teruo Fujii 《Biomicrofluidics》2012,6(4)
Water-in-oil microdroplets offer microreactors for compartmentalized biochemical reactions with high throughput. Recently, the combination with a sol-gel switch ability, using agarose-in-oil microdroplets, has increased the range of possible applications, allowing for example the capture of amplicons in the gel phase for the preservation of monoclonality during a PCR reaction. Here, we report a new method for generating such agarose-in-oil microdroplets on a microfluidic device, with minimized inlet dead volume, on-chip cooling, and in situ monitoring of biochemical reactions within the gelified microbeads. We used a flow-focusing microchannel network and successfully generated agarose microdroplets at room temperature using the “push-pull” method. This method consists in pushing the oil continuous phase only, while suction is applied to the device outlet. The agarose phase present at the inlet is thus aspirated in the device, and segmented in microdroplets. The cooling system consists of two copper wires embedded in the microfluidic device. The transition from agarose microdroplets to microbeads provides additional stability and facilitated manipulation. We demonstrate the potential of this method by performing on-chip a temperature-triggered DNA isothermal amplification in agarose microbeads. Our device thus provides a new way to generate microbeads with high throughput and no dead volume for biochemical applications. 相似文献
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Arun Ray Susri Ray Chaudhuri Biswajit Majumdar Sandip K. Bandyopadhyay 《Indian journal of clinical biochemistry : IJCB》2002,17(2):44-51
Oral administration of ethanol extract of the rhizome ofPirorhiza kurroa at a dose of 20mg/kg body weight, for 10 consecutive days, was found to enhance the rate of healing on Indomethacin-induced
gastric ulcer in rats, compared to the ulcerated group without treatment. The level of peroxidised lipid, in terms of thiobarbituric
acid reactive species (TBARS), in gastric tissue, was increased in ulcerated rats which was restored to near normalcy on treatment
with ethanol extract. The specific activity ofin vivo antioxidant enzymes, viz SOD and catalase and total tissue sulfhydryl (thiol) group, which were markedly decreased in ulcerated
group, were found to be significantly elevated (p<0.05), on treatment with the above extract, at the specified dose, compared
to the indomethacin—induced ulcerated group without any supporting treatment. The present study thus suggests that the ethanol
extract of rhizome ofPicrorhiza kurroa, at the dose of 20mg/kg body weight, accelerated the healing of stomach wall of indomethacin induced gastric ulcerated rats
by anin vivo free radical scavenging action. 相似文献
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Patra SK Jain A Sherwal BL Khanna A 《Indian journal of clinical biochemistry : IJCB》2010,25(3):315-318
To detect the site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-TB by DNA sequencing. 50 MDR-TB patients
were enrolled in our study after informed written consent. Mycobacterial DNA was extracted from sputum samples by Universal
Sample Processing (USP) method and RRDR of rpo B gene was amplified by PCR using primers RP4T and RP8T and then sequenced
by automated DNA sequencing. The nucleotide sequences of RRDR of rpo B gene were compared with the reference sequence. We
observed three different types of mutation in the RRDR of rpo B gene. The frequency of mutation in codon 531 (TCG → TTG),
526 (CAC → TAC) and 516 (GAC → GTC) are 60, 26.6 and 6.6% respectively. Of the total cases studied, 6.6% cases, although resistant
to rifampicin, did not show any mutation in the RRDR of rpo B gene. Codon 531 (TCG → TTG) is the most common site of mutation
in RRDR of rpo B gene for rifampicin resistance in MDR-pulmonary tuberculosis followed by codon 526 (CAC → TAC) and codon
516 (GAC → GTC). 相似文献
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D. Saravanakumar N. Elangeswaran S. Senthilkumar G. Vanaja S. Kamakshiammal C. Chandrasekar C. N. Deivanayagam Manjula Sritharan V. Sritharan 《Indian journal of clinical biochemistry : IJCB》2000,15(2):94-103
We have isolated and identified the biotype of environmental mycobacteria from the expectorate of leprosy patients, their
contacts, their drinking water supply and also from the sputa samples of tuberculosis patients. 78% of the isolates from lepromatous
leprosy patients and their contacts wereMycobacterium fortuitum- chelonae complex (MFC), 9%Mycobacterium avium complex (MAC), 9%Mycobacterium scrofulaceum and 4% wereMycobacterium smegmatis. Among the isolates from tuberculosis patients 63% belonged toM. fortuitum- chelonae complex, 19% toM. avium complex, 12% toMycobacterium Kansasii and 6% toM. smegmatis. All the isolates were multi-drug resistant when tested for sensitivity total of 21 drugs. TheMycobacterium fortuitum-chelonae complex organisms from leprosy contacts were more sensitive to rifampicin than those isolated from lepromatous leprosy and
tuberculosis patients. Among 23 isolates from leprosy patients one isolate was resistant to 20 drugs, one isolate to 17 drugs
and another isolate was resistant to 13 drugs. Among the 18 isolates from drinking water supply six showed resistance to more
than 12 drugs. Polymerase Chain Reaction (PCR) and subsequent hybridisation with specific probes confirmed all the isolated
strains as nontuberculous mycobacteria (Using genus primers and probe sensitivity 100%) and none asM. tuberculosis, suggesting that PCR could be used to rapidly identify mycobacteria at the genus level and to rule out tuberculosis in leprosy
patients at an early stage to decide on appropriate course of therapy. 相似文献
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A C-type mannose specific lectin (LRP) isolated from plasma of fresh water fishLabeo rohita showed 500 times higher specificity towards cell surface oligosaccharide (LAM) ofMycobacterium tuberculosis (H37Rv). Using biotinylated LRP, binding between lectin and LAM was demonstrated by ELISA and it was observed that even 3ng
of oligosaccharides might be detected using only 1μg of biotinylated lectin. 相似文献
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BackgroundAgkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market.ResultsThis method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species.ConclusionsThe proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.How to cite: Yingnuo L, Yanshuang W, Mingcheng Li, et al. Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene. Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.07.005 相似文献
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P. K. Manchanda H. K. Bid B. R. Achyut B. Mittal N Srivastava R. D. Mittal 《Indian journal of clinical biochemistry : IJCB》2007,22(1):61-64
The main adverse consequences of excess bodyweight are cardiovascular disease, type II diabetes, and several cancers, IL-1Ra
serum concentration has been reported earlier to increase in human obesity and it is therefore assumed that the polymorphism
of IL-1Ra may influence cytokine production. We designed this study to investigate whether the IL-1Ra polymorphism was associated
with obesity. A total number of 103 individuals; 19 lean (BMI<25 Kg/m2), 51 overweight (BMI 25–29.9 Kg/m2) and 33 obese (BMI≥30.0 Kg/m2) were enrolled in this study. Genotyping was performed using a polymerase chain reaction PCR amplification of the intron-2
fragment harboring a variable number of tandem repeat (VNTR) nucleotide sequences 86 pb of tandem repeat. The PCR products
were separated on 2% agarose gel. Statistical analysis was performed using SPSS software (version 11.5). We found no significant
difference in genotype and allele frequencies between the three groups; lean vs. overweight and lean vs. obese (p=0.323; 0.202;
0.123 and 0.068 resp). However, an increased risk for obesity had a propensity to be higher in those having genotype II/II.
This genotype has been reported to be a ‘high producer’ of IL-1Ra. Although no statistically significant relationship between
IL-1Ra polymorphism and BMI was observed, however, a trend towards an increase of allele*II in overweight and obese group was observed. This may suggest that IL-1Ra appears to be induced by inflammatory stimuli
as well as obesity-associated factors. This is relatively a pilot study: but nevertheless, may assist in identifying the pathophysiological
cause for obesity. 相似文献