Showing 20 results for Polymerase Chain Reaction
Volume 1, Issue 3 (10-2014)
Abstract
Background: Acne vulgaris is an inflammatory chronic disease of pilosebaceous unit. One of the most important factors playing a role in occurrence of acne is presence of Propionibacterium acnes. With the aim of molecular identification of the P. acnes from the acne vulgaris lesions, current research was carried out. Methods: In this study, contents within the lesions was collected from 70 patients. The presence of the P. acnes was examined by a specific PCR technique. Results: Of 70 samples, 58 samples (82.85%) were determined to be positive in terms of presence of P. acnes. No significant relationship was observed between presence of P. acnes and each one of the studied demographic factors, including gender, age, disease period, family background and treatment background. Conclusions: The adopted molecular technique has obviated the limitations associated with the culture method for identification of the bacteria. To overcome the problems with conventional culture techniques for P. acne, this PCR method is promising for better identification of this bacterium.
Volume 3, Issue 3 (9-2017)
Abstract
Background: Cryptosporidiosis is one of the most important parasitic diseases infecting a broad variety of animals and humans. In the present study, Nested PCR-RFLP-based assay was applied for genotyping of sheep cryptosporidiosis. The target of amplification was the 18S rRNA gene used to identify Cryptosporidium species
Materials and Methods: In the first step, 1300 faecal samples were collected from sheep in Tehran province, then the samples were examined for the presence of Cryptosporidium using modified acid fast staining. In the second step, DNA was extracted from the positive samples. Next, 18S rRNA gene was amplified by Nested-PCR in order to differentiate between the species. The PCR product was digested by Ssp1 restriction enzyme.
Results: Twenty two positive sheep samples were detected by modified acid fast method. The results were confirmed by molecular techniques. The 845 bp fragment of 18S rRNA was digested by restriction enzymes. Twenty samples showed a similar band on 2.5% agarose gel whereas 2 samples demonstrated different pattern. The sequences of two patterns indicated two species of C. andersoni and C. parvum.
Conclusion: In spite of other studies results introducing C. parvum as the major agent of cryptosporidiosis in sheep, in our study, C. andersoni was found to be dominant.
Volume 4, Issue 3 (9-2018)
Abstract
Aims: Transportation of clinical samples and long-term recoverability of fungal strains are critical to epidemiological studies. In addition, the study of fungi often requires the use of living pure cultures. The aim of this study was to evaluate the methods used to preserve culture collections of dermatophytes, consisted of storage in sterile distilled water, cryopreservation with glycerol, preserving in tryptic soy broth (TSB), and freezing mycobiotic agar.
Materials and Methods: in this experimental study, ninety-two dermatophyte isolates belonged to 10 species were tested. The freezing protocol was done in 4 forms of sterile distilled water, cryopreservation with glycerol, freezing mycobiotic agar, and preserving in TSB. The viability of the dermatophytes species was assessed after 3 years at morphological (macro and microscopic features), physiological (Using Dermatophyte Test Medium; DTM, urease test media, and the hair perforation test), and genetic levels by restriction fragment length polymorphism (RFLP).
Findings: The survival rate was 84 out of 92 water stored fungal strains (91.3%) and 81 out of 92 mycobiotic agar stored strains (88.0%) and 75 out of 92 glycerol 40% stored strains (81.5%) and 43 out of 92 TSB stored fungal strains (46.7%). Overall, more than 88% of the strains survived in the distilled water storage and freezing mycobiotic agar, methods, while storage in TSB had the least success in the maintenance of dermatophytes.
Conclusion: The procedure to preserve cultures in sterile distilled water is reliable, simple, and inexpensive.
Volume 4, Issue 3 (9-2018)
Abstract
Aims: Diagnosis of Listeria monocytogenes infections is critical for epidemiological study and prevention of diseases. This study aimed at identifying L. monocytogenes isolates, using Loop-Mediated Isothermal Amplification Method (LAMP).
Materials & Methods: Listeria strains were obtained from clinical and seafood specimen. All listeria strains were identified by standard microbiological and biochemical tests. The LAMP assay was performed at 65°C with a detection limit of 2.5 ng/μl for 46 min. Specific primers for the hylA gene were used to identify L. monocytogenes. The specificity of the assay was assessed, using DNA from L. monocytogenes ATCC 7644 and L. ivanovii ATCC 19119 and non-Listeria strains. Sensitivity of the LAMP assay was compared with polymerase chain reaction (PCR) method. Amplification LAMP products were visualized via calcein and manganous ions as well as agarose gel electrophoresis.
Findings: A total of 191 samples were obtained, including clinical and food samples. Then, 21 (10.9%) isolates were recovered from specimens. The LAMP results showed high sensitivity (97.2%) and specificity (100 %). The LAMP assay was higher sensitive than of the PCR assay.
Conclusion: This data showed that this method could be used as a sensitive, rapid, and simple identification tool for diagnosis of L. monocytogenes isolates and it may be suitable for epidemiological study plans.
Bahram Golestani, Afshane Jafari, Farokh Karimi,
Volume 7, Issue 2 (9-2016)
Abstract
Salmonella is the serious and prevalent bacteria that has important role in epidemic infections. Beside increase of antibiotics resistance in bacteria make that abundance researches did for introduction of replacement method for beard with bacteria infections. Copper NANO oxide particles are components that their anti-microbial nature be evidenced. In this research for discover of NANO particles probably mechanism on the genome of bacteria, salmonella selected as a model for Gram-negative bacteria. In this regard, at first the bacteria were treated with 30 and 60 µg/ml copper oxide NANO particles. At time intervals of 2, 4, and 24 hours. In this doses, bacteria was growth .So bacteria were treated with 90 and 120 µg/ml copper oxide NANO particles. In these doses growth of bacteria even after 24 h completely were stopped .Then their DNA were extracted. In order to investigate the effects of copper oxide NANO particles on the genome, the chain reaction techniques of (RAPD-PCR) was employed .Using the software NTSYS-PC, the results obtained from electrophoresis of PCR products on agarose gel were analysis. The results of the study revealed that copper oxide NANO particles not only affects the growth of bacteria but also affect the sequencing of genomic DNA and leads to the changes of them in different points.
Volume 8, Issue 3 (9-2022)
Abstract
Backgrounds: Abnormal vaginal discharge is a common problem among pregnant women. The most common cause of these discharges is bacterial vaginosis (BV), which has numerous complications and causes problems for pregnant mothers and their fetuses. The purpose of this study was to determine the BV frequency among pregnant women referring to a gynecology clinic in Arak city using Amsel and Nugent criteria, Alberta guideline, and PCR.
Materials & Methods: This descriptive study was performed on 70 vaginal samples of pregnant women in Arak to investigate the most common causes of vaginal discharge according to Amsel and Nugent criteria and polymerase chain reaction (PCR) method using specific primers targeted towards three bacteria: Gardnerella vaginalis, Atopobium vaginae, Mobiluncus curtisii. Data were analyzed using SPSS software and Chi-square test.
Findings: In this study, ten (14.28%) out of 70 pregnant women had positive bacterial vaginosis according to Amsel criteria. According to Nugent criteria and Alberta guideline, three (4.29%) cases were diagnosed with definite BV, 20 (32.26%) cases with intermediate BV with clue cells, 42 (67.74%) cases with intermediate BV without clue cells, and finally five (4.29%) cases with negative BV. Also, according to PCR, the frequency of G. vaginalis, M. curtisii, and A. vaginae in vaginal samples was 71.42% (50 cases), 64.28% (45 cases), and 30% (21 cases), respectively.
Conclusion: According to the obtained results, the prevalence of definite bacterial vaginosis was lower than that of vaginitis, and most patients suffered from nonspecific vaginitis.
S. Sheykhi , M. Amininasab, B. Saffari, S. Abdi,
Volume 9, Issue 1 (1-2018)
Abstract
Aims: Identifying the structure and function of alpha-Synuclein protein can lead to the development of appropriate treatments for Parkinson disease. The aim of the current study was to investigate DNA cloning and the expression of alpha-Synuclein protein in E. coli.
Materials and Methods: In this experimental study, the sequence of encoding alpha-Synuclein in pRK172 recombinant plasmid was amplified by Polymerase Chain Reaction (PCR), using best primers. The synthesized DNA was, then, digested by restriction enzymes and cloned into pET28a and recombinant plasmid was transferred into the expression strain of E. coli (BL21) by Calcium Chloride method. The expression of alpha-Synuclein gene was induced by Isopropyl-Beta-D-Thiogalactoside (IPTG) and the expression of alpha-Synuclein was investigated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) method. Sequencing was done, using the ClustalW algorithm by the BioEdit 5.0.9 program.
Findings: In products of DNA enzymatic digestive reactions and pET28a plasmid with restriction enzymes, the size of the fragments indicated the correctness of the enzymatic reactions. The synthesized DNA and pET28a plasmid were 407 and 5369 nucleotides, respectively. The translation of the sequence of the cloned fragment revealed a 100% similarity to the human alpha-Synuclein protein. In expressing the recombinant protein in comparison with negative control samples, adding IPTG increased the expression of alpha-Synuclein protein in all samples, especially 2 hours after induction. Most of alpha-Synuclein expressed from the pET28a-alpha-Synuclein plasmid accumulated in the bacteria as incorporated objects.
Conclusion: The alpha-Synuclein protein is cloned into the pET28a plasmid and formation of the objects incorporated by alpha-Synuclein is confirmed by the expression of the pET28a-alpha-Synuclein system and paves the way for producing this protein in high scale.
F. Karimi , E. Khodaie,
Volume 9, Issue 2 (9-2018)
Abstract
Aims: In recent years, according the benefits of chloroplast transformation, the cultivation of transplastomic plants and their products have been increased. Due to their biosafety concerns, their identification and labeling have become more widely considered. The aim of this study was to present an optimal method based on polymerase chain reaction (PCR) and nanobiosensor for detection of transplastomic tobacco plants and compare their sensitivity.
Materials and Methods: In the present experimental research, aadA gene as a chloroplast selectable marker was considered to design specific primer and probe. In PCR method, after optimization of aadA gene amplification, its sensitivity was evaluated with different percentages of transplastomic DNA. In nanobiosensor method at first, the labeled aadA probe was immobilized on graphene oxide (GO) and, then, hybridization reaction was optimized to identify target DNA sequence.
Findings: The amplification of 800 bp DNA related to aadA gene was observed. The PCR reaction allowed up to 5% DNA transplostomy tobacco to reproduce the aadA gene. In results of nanobiosensor after immobilization of aadA probe on GO, fluorescence emission was quenched and by adding the trasplastomic tobacco, DNA was observed again. In this method, up to 1% transplastomic tobacco DNA, fluorescence emission was significant in comparison with control tobacco plant.
Conclusion: The PCR method can detect a transplastomic tobacco plant with 5% DNA sensitivity and detect biomarker sensitivity with 1% DNA sensitivity.
The PCR method can detect a transplastomic tobacco plant with 5% DNA sensitivity and nanobiosensor can detect with 1% DNA sensitivity. Therefore, nanobiosensor method is not only a reliable diagnostic method, in addition to the PCR method for detecting transplastomic plants, but also has a higher sensitivity.
Volume 9, Issue 3 (10-2023)
Abstract
Background: This study aimed to compare the diagnostic efficacy of standard culture method with multiplex quantitative real-time polymerase chain reaction (qPCR) in examining cerebrospinal fluid (CSF) samples collected from patients with suspected meningitis.
Materials & Methods: A retrospective evaluation was conducted on 166 patients with suspected meningitis, who were treated in Vali-Asr hospital in Birjand, Iran between 2011 and 2020. Diagnosis of bacterial meningitis was based on CSF culture and multiplex qPCR results.
Findings: Among 166 patients, conventional methods identified causative pathogens in only 10.3% of cases, while multiplex qPCR detected pathogens in eight out of 25 culture-negative cases as well. The most common pathogens identified were enterovirus, Epstein-Barr virus, herpes simplex, Haemophilus influenzae, and Streptococcus pneumoniae.
Conclusion: Multiplex qPCR appears to be a more effective method than conventional culture in identifying bacterial and viral pathogens that most commonly cause meningitis. The incorporation of qPCR as a routine diagnostic method for meningitis in clinical practice could significantly enhance clinical decision-making and patient care.
M.s. Borhani , Z. Etemadifar , G. Emtiazi , E. Jorjani ,
Volume 9, Issue 4 (12-2018)
Abstract
Aims: Alkaline protease is one of the most important groups of industrial enzymes with many applications. The aim of this study was to determine the physicochemical parameters affecting the production of alkaline protease enzyme produced by Bacillus pseudofirmus MSB22 by one-factor-at-a-time (OFAT) method and optimize the production of this enzyme by the response surface methodology (RSM) in the form of a rotatable central composite design.
Materials and Methods: In the present experimental study, the isolation of microorganism producing alkaline protease from wastewater from sausage and lunch meat factories in Isfahan was carried out. The morphological and biochemical characteristics of the strain were performed according to the Bergey's book and amplification of 16S rRNA gene sequences. Detection of metalloproteinase gene and alkaline serine protease was done by polymerase chain reaction (PCR) reaction and enzyme activity measurement was performed by Folin reagent. Screening of variables effective in enzyme production was done, using one-factor-at-a-time method and optimization was performed by response surface methodology. MEGA 6 software was used for phylogenetic analyses. To analyze the data, the Design Expert 7 software and the one-way analysis of variance were used.
Findings: The maximum protease production, which was 1.85 times higher than that of OFAT method and 3.45 times higher than unoptimized conditions was obtained, using 1% w/v xylose, 3% w/v beef extract, 4% v/v inoculation size, pH 10, and 30°C. The established quadratic model had a great ability to predict responses to new observations due to a high value of the predicted determination coefficient.
Conclusion: OFAT and RSM strategies are useful screening and optimization methods, respectively and sub I and sub II genes (alkaline serine protease genes) are detected in Bacillus pseudofirmus MSB22.
Volume 10, Issue 1 (2-2024)
Abstract
Background: Gastroenteritis is the second leading cause of death worldwide, with a high prevalence in children. Among pathogenic microorganisms, viruses are one of the main causes of this disease. Thus, the aim of this research was to investigate the prevalence of diarrhea caused by human adenovirus (HAdV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) in children with hematological diseases for the first time in Iran.
Materials & Methods: This study was conducted on 120 stool samples stored in the clinical sample bank of the Cellular and Molecular Research Center of Qom University of Medical Sciences. These samples were obtained from immunocompromised children with gastrointestinal symptoms, who referred to one of the children's hospitals in Qom during 2018 to 2019. Genomes were extracted from the stool samples and evaluated using the polymerase chain reaction (PCR) method.
Findings: The prevalence of HAdV and EBV was reported in seven (5.8%) and one (0.8%) cases, respectively, and CMV was detected in none of the samples. No cases of co-infection were observed.
Conclusion: This study results show that there are diarrhea-causing viruses among patients in the study area. Fortunately, the prevalence of these infectious agents in patients with underlying conditions was relatively low. However, monitoring of these viruses in the feces of all patients, especially immunocompromised patients, is recommended.
Volume 10, Issue 4 (12-2024)
Abstract
Background: The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is a growing global public health concern due to the significant morbidity and mortality associated with infections caused by these bacteria. The main objective of this study was to determine the prevalence of class I integron in CRE isolates collected from patients in teaching hospitals affiliated to Mazandaran University of Medical Sciences (MAZUMS).
Materials & Methods: A total of 100 Enterobacteriaceae isolates were collected during March 2022 to March 2023 from MAZUMS teaching hospitals using a consecutive sampling technique. The isolates were distinguished using standard microbiological methods. The antibiotic resistance of the isolated strains to carbapenem was subsequently detected using antibiotic discs including imipenem and meropenem. Using the disc diffusion method, 73 carbapenem-resistant isolates were identified and subsequently investigated by genetic analysis using polymerase chain reaction (PCR).
Findings: Among the 73 carbapenem-resistant isolates, the most commonly found bacterial isolates were Klebsiella pneumoniae (39.72%), Escherichia coli (30.13%), and Serratia rubidaea (12.32%), respectively. Also, 100% of the isolates were resistant to meropenem, while these isolates showed lower resistance to imipenem (70%). Also, out of the 73 isolates, 64.38% were positive for the intI1 gene. K. pneumoniae isolates had the highest prevalence of the intI1 gene (89.65%).
Conclusion: The prevalence of class I integron among patients in MAZUMS educational hospitals is relatively high, exceeding 50%. Therefore, it is crucial to implement effective infection prevention measures and identify this gene in hospitals to hinder the rapid dissemination of these hazardous organisms.
Marziyeh Salehi Siavashani, Ruhollah Nakhai Sistani, Alireza Panahi,
Volume 13, Issue 2 (1-2023)
Abstract
Aim: Multiple sclerosis is important in Iran because of its high prevalence and low age of onset. It exerts a large burden on affected people and the health care system. Studies have shown that the genetic content of humans has a critical role in MS. The histocompatibility loci which their products present the foreign antigenic peptides for detection by lymphocytes are of MS associated genetic elements. In this study, the association of HLA-DQB1*03 with MS was studied in Tehran, using a PCR-based system.
Methods: In this case-control study, a total of 367 blood samples were collected from 172 patients and 195 healthy people. Both groups were similar in age and gender. Blood DNA was extracted, and PCR technique was used to identify the presence of the allele.
Results: In this study, the HLA-DQB1*03 allele in men was significantly higher than that of women (p = 0.002). Also, the allele was less frequent in patients than in healthy subjects (53% versus 67%), and this difference is significant (p = 0.02).
Conclusions: The DQB1*03 allele is significantly lower in patients with MS than in healthy people, and this relationship is more pronounced in male subjects. Therefore, it seems that this allele plays a protective role against MS disease.
Volume 15, Issue 79 (9-2018)
Abstract
Dairy products can be a rich source of diverse lactic acid bacteria (LAB) with high functional properties, such as exopolysaccharide (EPS) production. EPS are high molecular weight polymers that are composed of sugar units and are secreted by microorganisms into the surrounding environment. Produced EPS can be used as an additive by a health effect and texture properties. In this study, exopolysaccharide producing LAB (ropy and mucoid colonies) were isolated and identified from raw sheep milk and sheep yogurt that made in rural areas of Urmia. For this purpose, lactic acid bacteria cultured on MRS agar and M17 agar media and then isolated on the basis of the ability to produce exopolysaccharides to study the diversity of species by phenotypic methods (Gram stain, biochemical and physiological tests) and then identified by polymerase chain reaction (PCR). 7 strains of LAB isolated were Gram-positive as well as catalase negative and were able to produce large amounts of EPS. The amounts of bonded and abandoned exopolysaccharides which measured by phenol/sulfuric acid method were 40.28±0.2 to 65.26±0.47 mg/L and 105.68±3.2 to 136.35±0.2 mg/L, respectively. Sheep yogurt which manufactured traditionally in West Azerbaijan province contained exopolysaccharide-producing strains that can have the potential to use in the dairy industry.
Volume 16, Issue 89 (7-2019)
Abstract
Studying the microbiota of food materials is important from sanitary, spoilage and technological perspectives. Due to manual harvesting, grading and packaging of date fruit (Phoenix dactylifera L.), it's not surprising to find bacteria of human origin in date fruit products. On the other hand, microbes present in date palm, especially high moisture content fruits, could produce lactic acid and acetic acid and make the fruit sour, so this kind of microbes potentially could be useful especially in food fermentation and vinegar production. In this project, seven varieties of date fruits were examined chemically and microbiologically. Total solid and acidity of dates were between 72% to 86% and 0.06% to 0.78% respectively. Isolated bacteria were identified by 16S rDNA amplification and restriction analysis (ARDRA), sequencing and 16S rRNA gene comparison. The results show Staphylococcus epidermidis, Aerococcus, Bacillus and Leuconostoc mesenteroides are the present bacteria in local date fruits.
Volume 19, Issue 2 (9-2016)
Abstract
Introduction: Squamous cell carcinoma comprises approximately 94% of all oral cavities. One reasons for this cancer is Human Papilloma Virus (HPV) with different genotypes .Finding the most common genotypes will be helpful to control and prevent of spreading this cancer.
Materials and Method: 70 Paraffinated blocks were collected from cancer department of Imam Khomeini hospital in Tehran. All of these samples included histopathological report of dysplastic lesions .They weredeparaffinated.4 primers were designed for PCR .Then they were transferred to electrophoresis tank. Positive samples were sequenced by Mega 5.
Results: 8 HPV+ samples include of 3 HPV+6 and HPV+16 were found . HPV6 is the cause of genital warts that can spread by skin contact or oral- sexual behavior. 3 positive samples were found in women and the others were in men. (2% more in men) . People between 30 to45 are more sensitive for HPV than the other group. People up to 60 years old are sensitive too. All the samples were collected from different cities of Iran but most of the positive samples were found in Tehran and Islam Shahr.
Conclusion: These data confirm that HPV infection with high risk types (6, 16) could be one of the risk factors for oral cancer and it can spread by genital warts.
Volume 19, Issue 6 (11-2017)
Abstract
Quick and authentic identification of exotic and potentially invasive taxa with capability of causing high economic losses or detriments is essential prerequisite for effective plant quarantine and biological control initiatives. The order Thysanoptera includes several agricultural pest species that, not only because of their minute size but also due to their cryptic behavior, incline to undetected transport through international trade of plants. Identification of thrips, particularly at species level, is pretty demanding and requires expertise in knowledge about Thysanoptera. Moreover, in most cases, identification of larval Thysanoptera to species is impossible without presence of adults. Hence, there is a great desire for a facile, accurate, and highly reliable technique for thrips identification. The present study describes species-specific primers for four pest thrips species, and the use of a multiplex PCR assay to detect and to distinguish between the four target species. Five primers were used to simultaneously amplify a specific region of the mitochondrial DNA and produce species-specific fragments. Results indicated that the primers were capable of detecting these four species and amplifying uniquely sized, species-specific PCR products. Furthermore, using a multiplex PCR assay, the primers maintained specificity and sensitivity, and allowed detection of each of the four species in a single reaction. The stringency of the method was tested using specimens of different developmental stages and consistent results were obtained for all of the examined samples. This method is simple enough to be implemented by non-experts and also can be extended to any organism for which quick and reliable identification is needed.
Volume 20, Issue 8 (8-2020)
Abstract
Research on DNA is particularly important in the diagnosis, control, and treatment of many diseases, including cancer. Today, the use of digital droplet polymerase chain reaction (ddPCR) for different DNA tests has attracted much researchers’ attention. A large number of micron-sized droplets are required to perform ddPCR. In the present study, a ddPCR system was designed, fabricated, and evaluated using a microfluidic chip. The system comprises a microfluidic chip for droplet generation and a thermal cycling device needed for PCR. The droplet generation in the microchip was simulated in 3D. The simulation results were validated. The average error is about 5% in the radius of the droplets. The constructed thermal cycling device controls the chip temperature with a precision of ±1.5°C. The in-chip PCR process was successfully performed by applying 25 heat cycles. The fluorescent property was observed in most droplets that prove the thermal cycling device can provide the conditions for DNA proliferation in the laboratory. The images were processed, and different levels of fluorescent light were identified in the droplets. The coefficient of variation of the selected droplets is 2.5%, which gives a good accuracy compared to the acceptable amount for these types of systems (less than 8%). The results obtained from this fully native device can be used in many fields, including cancer detection, examination of malignant tissue, and evaluation of the success in tissue surgery.
Volume 21, Issue 7 (12-2019)
Abstract
Dairy factories produce high volume of sludge from bactofuge and separator. Meantime, global demand for the proteases is increasing. Recently, utilization and conversion of the waste materials into value added product is a sustainable process. The objective of this study was to investigate the potential of bactofuge and separator sludge to produce alkaline protease enzymes. Total viable aerobic and anaerobic counts were determined on Plate Count Agar at 37 and 50ºC for both types of sludge. Lactobacillus count in MRS Agar plates corresponded to 3.12±0.25 log CFU mL-1 for sludge of bactofuge and 3.085±0.2 log CFU mL-1 for sludge of separator. Mold and yeast had population levels of 2.3±0.1 log CFU mL-1 for bactofuge and 2.08±0.1 log CFU mL-1 for separator. Proteolytic bacteria were isolated from dairy sludge using Skim Milk Agar media. A clear zone of Skim Milk hydrolysis indicated protease-producing organisms. Different cultural parameters (temperature, pH, thermal shock, and kind of sludge) were optimized for maximal enzyme production. Maximum proteolytic activity was observed at 37◦C (P< 0.05). Isolated alkaline protease producing Bacilli were identified by Polymerase Chain Reaction (PCR). The species were identified as Bacillus cereus strain zk2, Bacillus sp. cp-h71, Bacillus thuringiensis strain ILBB224, and Bacillus sp. Bac6D2.
Volume 24, Issue 2 (2-2021)
Abstract
Introduction: Vibrio cholerae, the causative agent of cholera, has attracted a great deal of attention as one of the major causes of morbidity and mortality worldwide, especially in developing countries. In most laboratories, biochemical assays are primarily performed for possible detection of these strains, which are then followed by a PCR (polymerase chain reaction) test to verify their identity. This study aimed to optimize dot blot technique to detect Vibrio cholerae bacteria for V. cholera as an easy-to-use and beneficial method.
Methods: A dot blot hybridization test was developed in this study to identify V. cholerae isolates as well as to assess the sensitivity and specificity of this test compared whit biochemical and PCR tests routinely performed for V. cholerae screening and detection in clinical specimens.
Results: Herein, the dot blot hybridization test was optimized to detect V. cholerae. A combination of three biochemical assays and PCR test confirmed the results of dot blot hybridization test. This test was able to identify V. cholerae strains with a high sensitivity and specificity of 100%. Using the newly developed method, a set of 26 V. cholerae isolates collected from clinical samples were accurately identified.
Conclusion: This study optimized dot blot technique as a simple and useful assay that could be employed in V. cholerae monitoring programs and strategies to effectively detect V. cholerae strains in surface water and fecal specimens.
