Chloroform Method Research Articles

Efficiency Evaluation of DNA Isolation Techniques in Fins of Channa marulius Using PCR Amplification and NanoDrop

Highly pure and good-quality extracted DNA is vital for PCR amplification, gene sequencing, and species identification. Seven genomic DNA isolation techniques were used to isolate the DNA from the fins of Channa marulius. The concentration and purity of isolated DNA were assessed with PCR amplification of the mitochondrial COI gene and NanoDrop. The phenol-chloroform method was found significantly (P<0.05) higher in concentration (1001 ng.ul-1) as compared to all other six evaluated DNA isolation methods. In terms of purity of isolated DNA GeneJET, the Genomic DNA Purification Kit was found significantly (P<0.05) higher among all analyzed methods. Both the Phenol chloroform method and GeneJET Genomic DNA Purification Kit were successfully used in PCR amplification. Concluding that the Phenol chloroform method and GeneJET Genomic DNA Purification Kit may be successfully used in PCR amplification. The phenol-chloroform method and GeneJET Genomic DNA Purification Kit proved the best in the extraction of DNA in high concentration with maximum yield and purity from fins of C. marulius and can be helpful to isolate DNA from fish species because a small quantity of fin tissues is required for DNA isolation without harm to fish. Moreover, data reported from this study about DNA isolation techniques may be used for molecular approaches like PCR amplification and gene sequencing of fishes.

Improving the efficiency of DNA extraction from iron incrustations and oilfield-produced water

The quantity and quality of DNA isolated from environmental samples are crucial for getting robust high-throughput sequencing data commonly used for microbial community analysis. The differences in the nature and physicochemical properties of environmental samples impact DNA yields, and therefore, an optimisation of the protocols is always recommended. For instance, samples collected from corroded areas contain high concentrations of metals, salts, and hydrocarbons that can interfere with several steps of the DNA extraction protocols, thereby reducing yield and quality. In this study, we compared the efficiency of commercially available DNA extraction kits and laboratory-adopted methods for microbial community analysis of iron incrustations and oilfield-produced water samples. Modifications to the kits manufacturers’ protocols were included to maximise the yield and quality. For iron incrustations, the modified protocol for FastDNA Spin Kit for Soil yielded higher DNA and resulted in higher diversity, including the recovery of low-abundant and rare taxa in the samples, compared to DNeasy PowerSoil Pro Kit. The DNA extracted with modified phenol–chloroform methods yielded higher DNA but failed to pass quality control PCR for 16S sequencing with and without purification. The protocols mentioned here can be used to maximise DNA recovery from iron incrustations and oilfield-produced water samples.

DNA Extraction from Postmortem Blood: A Pilot Study for Advancing Molecular Diagnostics in Forensic Medicine Casework

Background: DNA’s role in forensic practice is widely acknowledged for its unparalleled accuracy in identification. While developed countries have established molecular autopsy programs as early as two decades ago, India is yet to initiate such a program. The isolation of DNA serves as the crucial first step in the molecular autopsy protocol. The postmortem blood sample is one of the good sources for DNA extraction which wasn’t considered with rigor by the scientific community so far.Aims and Objectives: The aim of this study was to investigate the specific time period within which DNA canbe effectively extracted from postmortem blood samples. The objective was to identify if there are patterns in the quality and purity of the extracted DNA based on the postmortem interval. Additionally, the study aimed to investigate if the cause of death influenced DNA extractability.Observation and Results: DNA can be extracted from postmortem blood within a timeframe of up to 72 hours after death, given that the deceased body was preserved in cold storage within 12 hours after death. Both the salting out method and the phenol-chloroform method yielded bands of comparable quality, with the phenol-chloroform method showing a slightly higher DNA yield. The average absorbance ratio was 1.4 for the salting out method and 1.6 for the phenol chloroform method, as determined using a Nanodrop.Conclusion: This study concluded that DNA extraction from postmortem blood samples is feasible within 72hours after death. The integrity of the DNA remained intact during this time, but the quality and purity gradually decreased as the postmortem interval increased. The cause of death did not significantly affect DNA extractability.

Molecular based identification and phylogenetic relationship by using cytochrome b gene of Pangasius pangasius.

Molecular appraoch for identification of unknown species by using Cytochrome b gene is an effective and reliable as compared with morphological based identification. For DNA barcoding universal molecular genes were used to identify the species. Cytochrome b is a specific gene used for identification purpose. DNA barcoding is a reliable and effective method compared to the different traditional morphological methods of specie identification. So,in the present study which was conducted to identify the species, a total of 50 fish samples were collected from five different sites. DNA was extracted by using the Phenol Chloroform method from muscle tissue. Five sequences were sequenced (one from each site), analyzed, and identified specific species as Pangasius pangasius. Identified sequences were variable in length from 369 bp (Site 1), 364 bp (Site 2), 364 bp (Site 3), 352 bp (Site 4), and 334 bp (Site 5). Identity matches on the NCBI database confirmed the specific specie as P. pangasius. A distancing tree was drawn to show maximum likelihood among the same and different species. Yet, in many cases fishes on diverse development stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative tool for species identification and phylogenetic study. This work intends to provide an updated and extensive overview on the DNA based methods for fish species identification by using Cytochrome b gene as targeted markers for identification purpose.

Association of ITPA 94C>A genetic polymorphisms with azathioprine induced adverse effects in the South Indian population.

Azathioprine (AZA) is an effective immunosuppressant commonly used for malignancy and immune-mediated disorders. The association between genetic polymorphisms and AZA-induced adverse effects has not been elucidated. Hence this study aimed to evaluate the relationship between single nucleotide polymorphisms of ITPA (C94A) with azathioprine-induced adverse effects. A cross-sectional study was performed on 120 patients who were on AZA therapy for immunobullous disorders and inflammatory bowel disease (IBD). Eligible patients were enrolled from outpatient Departments of dermatology and medical gastroenterology and five mL ofblood was collected after obtaining written informed consent. DNA extraction and genotyping were done by phenol-chloroform method and real-time polymerase chain reaction (RT-PCR), respectively. The minor allele frequency of ITPA (A allele) was 30.8 %. The mutant genotypes of ITPA (C94A) were found to have no significant association with overall adverse effects in the South Indian patients on AZA therapy. We report no significant association between ITPA rs1127354 genetic polymorphism and adverse effects in the South Indian patients on AZA therapy.

Biotransformation of starch-based wastewater into bioplastics: Optimization of poly(3-hydroxybutyrate) production by Cupriavidus necator DSM 545 using potato wastewater hydrolysate

Biodegradable biopolymers, such as polyhydroxyalkanoates (PHAs), have emerged as an alternative to petrochemical-based plastics. The present work explores the production of PHAs based on the biotransformation of potato processing wastewater and addresses two different strategies for PHA recovery. To this end, culture conditions for PHA synthesis by Cupriavidus necator DSM 545 were optimized on a laboratory scale using a response surface methodology-based experimental design. Optimal conditions rendered a PHB, poly(3-hydroxybutyrate), accumulation of 83.74 ± 2.37 % (5.1 ± 0.2 gL−1), a 1.4-fold increase compared to the initial conditions. Moreover, polymer extraction with non-halogenated agent improved PHB recovery compared to chloroform method (PHB yield up to 78.78 ± 0.57 %), while maintaining PHB purity. (99.83 ± 4.95 %). Overall, the present work demonstrated the potential valorization of starch-based wastewater by biotransformation into PHBs, a high value-added product, and showed that recovery approaches more eco-friendly than the traditional treatments could be applied to PHB recovery to some extent.

Comparative study of extraction and analysis of DNA from different temperatures using phenol chloroform method

The extraction of DNA from biological samples is a fundamental process in forensic investigations, providing invaluable information for the identification and profiling of individuals involved in criminal activities. Saliva is a commonly encountered biological fluid at crime scenes and can serve as a rich source of DNA evidence. This article focuses on the extraction of DNA from saliva samples and explores the methods and techniques employed in forensic investigations to optimize DNA recovery and maximize profiling success. Saliva samples were collected in saline in a swab tube and half of the samples were kept at 4C and half at Room Temperature (RT) to understand the temperature variance. After that, DNA extraction was done using the phenol-chloroform method. The quality and quantity of the extracted DNA are assessed using established techniques such as Gel electrophoresis and qPCR amplification. The results of the experiments reveal the efficiency and reliability of the DNA extraction method for saliva samples. DNA was successfully extracted from the set conditions. Although the DNA bands obtained from 4oC were more prominent compared to the samples kept at RT. The identification of optimal DNA extraction methods for saliva samples can contribute to the development of standardized protocols and guidelines for forensic DNA analysis. In conclusion, this article sheds light on the extraction of DNA from saliva samples for its use in forensic investigation. The outcomes of this study have the potential to improve the efficiency and effectiveness of forensic investigations. Further research in this field is warranted to explore emerging technologies and refine existing methodologies for DNA extraction from saliva samples in forensic contexts.

Isolation and whole genome sequencing of a lipophilic anaerobic bacterium, a representative of the species complex <i>Corynebacterium tuberculostearicum</i>, from a tuberculosis focus

Background. The study of the lower respiratory tract microbiome has been actively developed inrecent years with the help of whole genome sequencing (WGS) methods. Due to this, it became clear that the nature of the lungs microbiota is very different from other microbial communities inhabiting the human body. One of the important directions in the study of pathological lungs biocenosis is the study of the role of the satellite microbiota of the tuberculosis focus. The aim of the work. To isolate and characterize oxygen-tolerant anaerobes from the necrotic contents of tuberculomas. Materials and methods. Biopsy material from 5 patients with pulmonary tuberculosis was obtained during a planned surgical treatment of tuberculoma. A pure culture was isolated from one sample during anaerobic cultivation. Lipase activity of strain was determined by plating on brain heart infusion agar (HIMEDIA, India) supplemented with 0.1 % Tween-80 and 10 mM of CaCl2. Antibiotic susceptibility was determined by RAPMYCO and SLOWMYCO of TREK Diagnostic Systems (Thermo Fisher Scientific, USA). DNA from the sediment of the broth culture was isolated by the CTAB chloroform method. Whole genome sequencing was performed on a DNBSeq-G400 NGS sequencer by Genomed (Russia). Results. Based on WGS results and phylogenetic analysis, the strain was identified as Corynebacterium kefirresidentii. The strain was characterized by high lipase activity and resistance only to Isoniazid, Ethionamide and Trimethoprim/Sulfamethoxazolin. Conclusion. The isolation of a lipophilic anaerobic representative of the Corynebacterium tuberculostearicum species complex from a tuberculous focus indicates a possible role of the non-tuberculous microbiota in the liquefaction of caseous necrosis. We assumed that in some cases, favorable conditions are created inside the tuberculous focus for the development of satellite anaerobic lipophilic microbiota.

Efficacy of cell-free DNA as a diagnostic biomarker in breast cancer patients

Breast cancer is the most prevalent and leading cause of mortality worldwide among women. Cell-free DNA (cfDNA) analysis is an alternative quantitative approach to conventional methods for cancer diagnosis. The current research project aimed to determine the efficacy of cfDNA as a diagnostic biomarker in breast cancer patients in Pakistan. Eighty-four female breast cancer patients were selected as cases, and 152 healthy females as controls. Immunohistochemistry was performed to identify tumor biomarkers along with clinical profiling. cfDNA was extracted from serum using the phenol–chloroform method. The cfDNA level in the serum was estimated using Agarose Gel Electrophoresis and Nanodrop. SPPS version 25.0 was used to perform statistical analyses. The results showed that the cancer biomarkers were significantly associated with breast cancer. The changes in hematological parameters were insignificant, whereas the biochemical parameter variations between the cases and controls were statistically significant. A significant association of cfDNA level with breast cancer was observed. Further cfDNA levels and cancer biomarkers were not statistically significant. A significant correlation was observed between cfDNA and biochemical parameters, except for creatinine, whereas hematological parameters showed no significant correlation.ROC analysis declared cfDNA as an authentic diagnostic marker for breast cancer. It was concluded that the level of cfDNA is significantly increased in breast cancer patients and can be utilized as a diagnostic biomarker.

Optimising faecal sample storage and DNA extraction procedures to help the implementation of forest elephant conservation strategies

AbstractFaecal samples are an important source of genetic information for studies of wild animals. The quality and quantity of faecal DNA can, however, be affected by different factors. Our goal was to compare different dung sample storage and DNA extraction methods to optimise the quality and quantity of DNA extracted from forest elephant dung samples. Dung samples (n = 132) of forest elephants were collected in a forest area in Côte d'Ivoire. Each faecal sample was preserved in 50 mL cryotubes using five different methods: Ethanol 70%, formalin 10%, Sodium acetate, acetic acid, and formalin (SAF), dimethyl sulfoxide, and Sodium chloride (20% DMSO), and silica gel. We used the Cetyl Trimethyl Ammonium Bromide (CTAB) cationic detergent and the phenol chloroform DNA extraction procedures for comparison. DNA was successfully obtained from all 132 faecal samples using both DNA extraction methods. Regarding the total DNA extracted independently from the storage buffers, the phenol chloroform method yields a mean DNA concentration (2163.93 ± 918.62 ng/μL) sevenfold higher than that obtained with the optimised CTAB method (338.15 ± 528.54 ng/μL). Our study indicates optimised faecal sample storage and DNA extraction procedures that can help implement conservation strategies for forest elephants.

First Report of Tar Spot on Corn Caused by Phyllachora maydis in the Great Plains.

Tar spot caused by the fungus Phyllachora maydis Maubl. is a significant foliar disease of corn (Zea mays L.). Threatening corn production across the Americas, this disease can reduce the quality of silage and grain yield (Rocco da Silva et al. 2021; Valle-Torres et al. 2020). Lesions caused by P. maydis are usually black, glossy, and raised stromata on the leaf surface and occasionally on the husk. (Liu 1973; Rocco da Silva et al. 2021). Samples consistent with tar spot of corn were collected between September and October of 2022 from 6 fields in Kansas, 23 in Nebraska, and 6 in South Dakota. One sample was selected from each of the three states for further microscopic evaluation and molecular analysis. Signs of the fungus were visually and microscopically confirmed in eight Nebraska counties in October 2021; however, in the 2021, season tar spot sings were not found in Kansas and South Dakota. In the 2022 season disease severity varied by location; some fields in Kansas had <1% incidence, whereas in South Dakota disease incidence approached 1-2%, and in Nebraska between <1-5%. Stromata were present on both green and senescing tissues. Morphological characteristics of the pathogen were similar and consistent with the description of P. maydis (Parbery 1967) from all examined leaves across all locations. Asexual spores (conidia) were produced in pycnidial fruiting bodies ranging in size 1.29 to 2.82 x 8.84 to 16.95 µm (n = 40, average 1.98 × 13.30 μm). The pycnidial fruiting bodies were often found adjacent to perithecia within the stromata. For molecular confirmation, stromata were aseptically removed from leaves collected at each location and DNA extracted using a phenol chloroform method. The internal transcribed spacer (ITS) regions of the ribosomal RNA gene were sequenced using ITS1/ITS4 universal primers (Larena et al. 1999). Amplicons were Sanger sequenced (Genewiz, Inc., South Plainfield, NJ), and a consensus sequence for each sample was deposited in GenBank: Kansas (OQ200487), Nebraska (OQ200488), and South Dakota (OQ200489). Using the BLASTn, sequences from Kansas, Nebraska and South Dakota showed 100% homology with 100% query cover to other P. maydis GenBank accessions (MG881848.1; OL342916.1; OL342915.1). Koch's postulates were not performed given the obligate nature of the pathogen (Muller and Samuels 1984). This report documents the first confirmation of tar spot on corn in Kansas, Nebraska, and South Dakota (Great Plains).

Screening of Biological Activity of Selected Medicinal Orchids of Western Ghats, Karnataka, India

The present work was carried out to investigate the presence of bioactive compounds in selected medicinal orchids such as Dendrobium jerdonianum, Dendrobium barbatulum, Eria mysorensis, Bulbophyllum neilgherrense, and Pholidota pallida. The phytochemical, antibacterial, and antioxidant properties of the plants were evaluated using extracts obtained through chloroform, methanol, and aqueous extraction methods, respectively. The results of the phytochemical test showed the presence of diverse classes of secondary metabolites such as alkaloids, steroids, triterpenes, saponins, tannins, flavonoids, carbohydrates, resins, proteins, glycosides, and phenolics. The presence of these phytochemicals varied among different solvent extracts among the selected plants. Among the plant extracts tested, D. Jerdonianum and P. pallida offered significant p ≥ 0.05 antibacterial properties against Escherichia coli, Salmonella typhi, Bacillus subtilis, Shigella flexneri, and Pseudomonas aeruginosa with a minimum inhibitory concentration (MIC) ranging from 0.312 to 2.5 mg/mL. Among the plant extracts tested, methanol extract of Erythrina mysorensis (29.38%), B. neilgherrense (15.92%), P. pallida (13.84%), and D. barbatulum (13.10%) showed highest phenolic contents, followed by chloroform and aqueous extracts in all the plants. E. mysoriensis and D. jerdonianum exhibited highest antioxidant properties at 85.41% and 81.28% (with IC50 values of 36.49 and 43.72 μg/mL), respectively, while the positive control, gallic acid, displayed antioxidant properties of 94.61% (with an IC50 values of 30.49 μg/mL). These results warrant further studies on using these selected orchids for therapeutic and pharmaceutical applications.

Evaluation of Adulteration, Caffeine Content, and Antioxidant Activity in Different Black Tea Brands

Tea established as a refreshing stimulant beverage rich in caffeine, sugars, lipids, and aromatic substances demonstrates a wide variation in the quality depending on the type of brand. In this study, five different black tea market brand samples collected from the local market were evaluated for adulterants, caffeine content, and antioxidant activity. Based on the laboratory tests, artificial colors, azo colours, and sand was detected in 80% of tea samples. Caffeine content was determined by the chloroform method. The tea samples exhibited a wide range of antioxidant activity (3.29±1.03 to 15.96±1.2) assayed by the DPPH free radical scavenging method. These investigations could go a long way in promoting the production of quality foodstuff in the larger interest of consumers.

Comparison of Modified Manual Acid-Phenol Chloroform Method and Commercial RNA Extraction Kits for Resource Limited Laboratories.

In a comparative experimental cross-sectional study, RNA was extracted from oral swabs and blood samples from 25 healthy individuals at the Department of Molecular Medicine, KNUST. RNA was extracted by the manual AGPC extraction method and commercial RNA extraction kits. The quantity (ng/μl) and purities (260/280 nm) of the extracted RNA were measured spectrophotometrically using the IMPLEN NanoPhotometer® N60. The presence of RNA in the extracts was confirmed using 2% agarose gel electrophoresis. Statistical analyses were conducted using R language. The yield of RNA extracted from blood and oral swab samples using modified AGPC was significantly higher compared to the commercial methods (p < 0.0001). However, the purity of RNA extracted by the manual AGPC method from blood was significantly lower than the commercial methods (p < 0.0001). Moreover, the purity from oral swabs using the manual AGPC method was significantly lower compared to QIAamp (p < 0.0001) and the OxGEn kits method (p < 0.001). The modified manual AGPC method has a very high yield of RNA extracts using blood samples, which could serve as an alternate cost-effective method for RNA extraction in resource-limited laboratories; however, its purity may not be suitable for downstream processes. Moreover, the manual AGPC method may not be suitable for extracting RNA from oral swab samples. Future investigation is needed to improve the purity of the manual AGPC RNA extraction method and also confirmation of the obtained results by PCR amplification and RNA purity verification by sequencing.

The phylogenetic authentication of Ophiocordyceps sphecocephala from Lang Biang Biosphere Reserve, Lam Dong, Vietnam

During our expedition at Lang Biang Biosphere Reserve, samples of DL0023, parasitizing on wasps, were collected, then sent to the biology laboratory, Dalat University, Lam Dong Province, Vietnam, to preliminarily morphologically identify the name of this species as Ophiocordyceps sphecocephala. The method of Phenol (pH = 8)/Chloroform method was applied to isolated DNA. The PCR assays and sequencing were performed to amplify and sequencing ITS, nrLSU, and nrSSU, respectively. The phylogenetic methods of neighbor-joining, maximum likelihood, and maximum parsimony were constructed to identify the relationship between DL0023 and referent sequences. We successfully amplified and sequenced the target genes, and constructed concatenated data containing 29, 33, and 30 sequences of ITS, nrLSU and nrSSU, respectively, belonging to Clavicipitaceae clade A, clade B and clade C, and 02 sequences belonged to Glomerellaceae. The results of phylogenetic analysis shown that DL0023 was accepted at Ophiocordyceps and strongly formed the monophyletic group Ophiocordyceps sphecocephala with a high supported value. In conclusion, the concatenated dataset’s phylogenetic analyses effectively supported the identification of DL0023 as Ophiocordyceps sphecocephala.

Detection for Suspected Genetically Modified Maize and Soybean Crops in the Selected Places of Ethiopia

Detection of genetically modified organisms (GMOs) in crops is an important issue for all the subjects involved in seed quality control and customer’s right. Due to the increasing number of GMOs research and development activity in the globe during the past few years, it has become necessary to screen and regulate highly practiced GMO crops. In this study soybean and maize crops samples were collected at different agricultural farm lands and commercial super markets that suspected for GMOs. Then, genomic DNA were extracted from crops samples by using Phenol chloroform method. By following ,the extracted genomic DNA amplified by using two oligonucleotide primer sequences such as CaMV35s and Lectin with the help of thermal cycler Polymerase Chain Reaction (PCR). In addition, positive control taxon -specific Invertase and Lectine genes for maize and soybean were used respectively. Finally, PCR amplicons were run on horizontal agarose gel electrophoresis for the confirmation of amplification. After that electrophoresis results were observed under gel documentation system. Invertase and Lectine gene events were detected in all maize and soybean samples, respectively. However, fortunately in all collected samples of maize and soybean in different places of the country the suspected foreign genes were not detected. Finally,this research result concludes that in the studied area soybean and maize crop samples are GMO free.

Genetic polymorphism of the BMP15 gene in West African sheep (Fulani) breed in Sudan

Genetic polymorphism studies in domestic animals aim at assessing genetic variations within and across breeds mainly for preservation purposes. The bone morphogenetic protein 15 (BMP 15) gene is a growth factor and a member of the transforming growth factor (TGFβ) superfamily specifically expressed in oocytes. One of the very exciting and perhaps crucial actions of BMP-15 is its ability to inhibit Follicle Stimulating Hormone (FSH) receptor expression. The West African sheep originated in the West African republics. Three strains were named after a person (Omer Uda) conveyed to Sudan in 1930. These breeds include (Uda, Sankara, and Weila) strains. They are habitually raised to gather as mixed pastoral flocks and trailed altered paths during the summer and autumn. Genetic polymorphism of the BMP 15 gene in Sudanese sheep is not reported. Consequently, this work aimed to study the genetic polymorphism in BMP-15 in West African sheep breeds. Blood samples were taken from thirty ewes of West African sheep. DNA samples were isolated from blood samples using the Guanidine chloroform method, and the quality of the DNA was checked on 2% agarose gel electrophoresis. Mutation in the BMP15 gene was screened using the forced PCR-RFLP technique. The fundamental discovery of the present study was the nonexistence of polymorphism at the BMP15 gene in West African sheep. The nucleotide sequence of the PCR product (141 bp) revealed that the C nucleotide has not changed to T at this locus (C-T) of the BMP15 gene in all samples.

Variation in soil organic carbon content and its dynamics under different cropping sequences and nutrient management practices in middle indo-gangetic plains

In the era when the population is escalating at a rapid rate while on other hand the land for cultivation is declining, there is tremendous pressure in high fertile lands like the Indo-Gangetic plains to produce food grains with intensified cropping. This intensification of agriculture has lead to stagnation of yield in cropping systems due to decline in soil organic matter and nutrient cycling. Soil organic carbon along with the microbial biomass are important indicators of physical, chemical as well as biological properties. This two parameters can be determined by various methods like dehydrogenase activity, soil respiration rate and microbial biomass calculation by chloroform and irradiation method. The current paper comprises of the study of different cropping systems in the Indo Gangetic plains in order to analyze its different organic carbon pool, relationship between them and different land use system that influences them. It was noticed that the system receiving both organic and inorganic fertilizers had higher total organic carbon, higher respiration rate, labile carbon along with higher microbial population. Under the nutrient management consisting of FYM + NPK fertilizer treatment there was increasing microbial biomass carbon and total organic carbon over other treatments because of more or higher root biomass. Therefore for a healthy soil and sustainable production system in high fertile and demand area of Indo Gangetic plain it is necessary to assess the soil organic carbon pools and maintain them by using integrated nutrient management.

Detection and species determination of malaria parasites by microscopy, antigen detection, polymerase chain reaction, and loop-mediated isothermal amplification assay in a tertiary care hospital

Background: Malaria is a major health issue in tropical and subtropical areas. Out of all subtypes, Plasmodium falciparum (Pf) is the most dangerous form accounting for high mortality and morbidity. It is transmitted by infected female anopheles mosquitoes and infected blood transfusions. Aims and Objectives: The aim of the study is to establish correct diagnosis by direct microscopy, Immunochromatographic test (ICT), and molecular studies. Materials and Methods: This prospective study was conducted in the PG Department of Microbiology, SCB Medical College, Cuttack. Thick blood smears were drawn and then stained with Leishman’s stain to visualize falciparum rings. DNA was extracted from infected blood samples by phenol chloroform method with some modification as described by Sambrook and Russel for molecular analysis. Results: In the present study, 150 cases of malaria were analyzed. The male: female ratio was 1.7:1 and age ranged from 0 to 56 years. The Plasmodium vivax positivity was compared with thin smear to 21 (84%) in ICT, 100% both polymerase chain reaction (PCR) and loop mediated isothermal amplification assay (LAMP) assays followed by the Pf positivity as 76 (92.7%) in ICT, 82 (100%) both PCR and LAMP assays, respectively. The results obtained were statistically significant with P &lt; 0.001. The PCR and LAMP showed 100% response to specificity and positive predictive value. Conclusion: The present study established the role of molecular tests such as PCR and LAMP are highly specific for diagnosis of Plasmodium species whereas they are more or less similar in sensitivity as compared to other diagnostic methods such as ICT and microscopy.

Detection of Leishmania donovani using ITS1-RFLP from positive and negative smear samples among clinically reported patients visiting University of Gondar Comprehensive Specialized Hospital

BackgroundVisceral leishmaniasis is caused by the Leishmania donovani species complex that can spread to internal organs and leading to death if not treated on time. Diagnosis of leishmaniasis is based on clinical signs and symptoms, microscopy, serological and molecular techniques. Because of a broad spectrum of diverse clinical manifestations and similarities of the responses to different species, identification to the species level is often difficult for the proper patient treatment and management. Therefore, the objective of this study was to evaluate the PCR- RFLP assay of the ITS1 region for identification of L. donovani species from clinical smear slide patient samples.MethodDNA extraction was performed on a total of 90 smear slide samples using phenol—chloroform method. The PCR detection limit was determined by L. donovani reference strain DNA. The ITS1 region was amplified at 320 bp using LITSR/L5.8S genus specific primers and then the ITS1-PCR products were subjected to RFLP assay for confirmation of L. donovani species using HaeIII restriction enzyme.ResultsOf the total samples ITS1-PCR revealed the true positive, false positive, true negative, and false negative results of 42 (46.7%), 6 (6.7%), 37 (41.1%) and 5 (5.6%), respectively. Considering microscopy as the gold standard, the sensitivity, specificity, positive predictive values, and negative predictive values of the ITS1- PCR technique was 89.4%, 86.0%, 87.5%, and 88.1% respectively. All ITS1-PCR positive clinical samples were confirmed as L. donovani species by PCR–RFLP patterns.ConclusionIn conclusion, the ITS1- RFLP method is highly sensitive and more specific for identification of L. donovani species in the smear negative clinical samples of visceral leishmaniasis patients. There is also significant association and degree of agreement between the two methods. For direct identification of L. donovani species from clinical samples, irrespective of genus and species level, PCR–RFLP is more recommendable than a microscope.