Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea

2020 ◽  
Author(s):  
Sanjeev Kumar ◽  
Jagan Mohanarao Gali ◽  
T.K. Dutta ◽  
P. Roychoudhury ◽  
P.K. Subudhi
Keyword(s):  
Bacterial Species ◽  
False Negative ◽  
Acute Diarrhoea ◽  
Lamp Assay ◽  
Dna Amplification ◽  
Analytical Sensitivity ◽  
Conventional Pcr ◽  
Isolation And Identification ◽  
Reliable Technique ◽  
E Coli

Background: Diarroeagenic Escherichia coli (DEC) including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene is considered as marker for EPEC and EHEC. Laboratory diagnosis of such infections is carried out by traditional bacteriological techniques and by conventional PCR assays. Those techniques often provide false negative result and at the same time are costly as well as difficult to perform in the field level. The loop-mediated isothermal amplification (LAMP) is a new generation DNA amplification assay is developed for detection of eaeA gene in E. coli isolated from diarrhoeic piglets.Methods: Samples were collected from diarrhoeic piglets for isolation and identification of E. coli. eaeA gene was detected by conventional PCR using specific primers in all the isolates. LAMP assay was standardized for detection of eaeA gene. Analytical sensitivity of LAMP was evaluated using 10 fold serially diluted E. coli genomic DNA. The specificity of the LAMP assay was determined by evaluating the cross reactivity with 19 other enteric and non-enteric bacterial species. Standardized LAMP was applied for detection of eaeA gene in the field isolates.Result: A total of 37 (24.67%) isolates were recorded as positive for eaeA gene by conventional PCR, while 49 (32.67%) isolates were recorded as positive for eaeA gene by LAMP assay. The LAMP assay was 10 times more sensitive than conventional PCR. LAMP assay was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR, which can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.

Development of a LAMP assay for rapid detection of different intimin variants of attaching and effacing microbial pathogens

2013 ◽  
Vol 62 (11) ◽  
pp. 1665-1672 ◽  
Author(s):  
Zhang Xue-han ◽  
Ye Qing ◽  
Liu Ya-dong ◽  
Li Bin ◽  
Ivanek Renata ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Microbial Pathogens ◽  
Conventional Pcr ◽  
Cell Adherence ◽  
E Coli ◽  
Faecal Samples ◽  
Specialized Equipment ◽  
Field Setting ◽  
Online Software

Intimin harboured by pathogenic Escherichia coli (E. coli) strains is a key virulence factor involved in host cell adherence and colonization. Twenty-seven intimin-encoding E. coli attaching and effacing (eae) gene variants have been reported according to their 3′ binding domain sequences. In our study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay to detect all known intimin variants. Four primers specific for six regions of eae genes were designed using online software. The eae-LAMP assay was highly specific and detected all 27 tested eae variants; no cross-reactions were observed with genes from enterotoxigenic E. coli (ETEC), E. coli BL21, Salmonella, Shigella, Listeria monocytogenes, or Streptococcus suis type 2 (SS2). With the lowest detection limit of approximately 10 copies per reaction the eae-LAMP assay was 100 times more sensitive than conventional PCR. These results, and the results of tests involving food and faecal samples artificially contaminated with E. coli O157 : H7 (eaeγ+), show that the eae-LAMP assay is a simple, rapid, sensitive and specific tool for detecting intimin variants from pathogenic strains of E. coli. The eae-LAMP assay has great potential for wider applications, not only in the laboratory but also in the field setting, as it does not require specialized equipment.

scholarly journals Rapid detection of cytochromecd1-containing nitrite reductase encoding genenirSwith loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
Qianqian Yang ◽  
Xuzhi Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification

AbstractThe cytochromecd1-containing nitrite reductase,nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance ofnirSis a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a new molecular biology technique termed loop-mediated isothermal amplification (LAMP) was developed to rapidly detectnirSgene using those ofPseudomonas aeruginosato optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other bacterial species lackingnirSgene. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, a cell-template based LAMP assay was also developed for detectingnirSgene that directly used bacterial cells as template rather than genomic DNA. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success, and bulky and sophisticated equipment were not needed. Further, thenirSgene ofP. aeruginosain spiked seawater samples could be detected with both the DNA-template based LAMP assay and the cell-template based LAMP assay, thereby demonstrating the practicality of in-field use of them. In summary, the LAMP assays described here represent a rapid, user-friendly, and cost-effective alternative to conventional PCR.

scholarly journals Unveiling the Virulent Genotype and Unusual Biochemical Behavior of Escherichia coli ST59.

2021 ◽  
Author(s):  
Ana Carolina de Mello Santos ◽  
Bruna Fuga ◽  
Fernanda Esposito ◽  
Brenda Cardoso ◽  
Fernanda Fernandes Santos ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Genomic Analysis ◽  
Epidemiological Studies ◽  
Sequence Type ◽  
Automatic Identification ◽  
Biochemical Tests ◽  
Isolation And Identification ◽  
E Coli ◽  
Metabolic Trait ◽  
Virulence Markers

Extraintestinal pathogenic E. coli (ExPEC) is a leading cause of human and animal infections worldwide. The utilization of selective and differential media to facilitate the isolation and identification of E. coli from complex samples as water, food, sediment, and the gut is common in epidemiological studies. During a surveillance study, we identified an E. coli strain isolated from human blood culture that displayed atypical light cream-colored colonies in chromogenic agar, being unable to produce β-glucuronidase and β-galactosidase enzymes in biochemical tests. Genomic analysis showed that the strain belongs to the sequence type ST59 and phylogroup F. The evaluation in silico of 104 available sequenced lineages of ST59 complex showed that most of them belong to serotype O1:K1:H7, are β-glucuronidase-negative, and harbor a virulent genotype associated with the presence of important virulence markers such as pap , kpsE , chuA , fyuA , and yfcV . Most of them were isolated from extraintestinal human infections in diverse countries worldwide and could be clustered/subgrouped based on papAF allele analysis. Considering that all analyzed strains harbor a virulent genotype, and most do not present the typical biochemical behavior of the E. coli species, we alert that they could be misclassified or underestimated, especially in epidemiological studies where the screening criteria rely only on typical biochemical phenotypes as happens when chromogenic media are used. Importance The usage of selective and differential media is a rule that guides presumptive bacterial identification based on specific metabolic traits that are specific to each bacterial species. When a bacterial specimen displays an unusual phenotype in these media, this characteristic may drive to bacterial misidentification or a significant delay in its identification, putting a patient at risk depending on the infection’s type. In the present work, we describe a virulent E. coli sequence type (ST59) that does not produce the beta-glucuronidase enzyme (GUS-negative), which is the metabolic trait widely used for E. coli presumptive identification in diverse differential media. The recognition of this unusual metabolic trait may help in the proper identification of ST59 isolates, the identification of their reservoir, and the evaluation of the frequency of these pathogens in places where automatic identification methodologies are not available.

scholarly journals Optimization of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Schistosoma Mansoni (Trematoda: Digenea) Detection in Biomphalaria spp. from Endemic Areas for Schistosomiasis in Brazil

2021 ◽  
Author(s):  
Silvia Gonçalves Mesquita ◽  
Floria Gabriela dos Santos Neves ◽  
Ronaldo Guilherme Carvalho Scholte ◽  
Omar dos Santos Carvalho ◽  
Cristina Toscano Fonseca ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
False Negative ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Conventional Pcr ◽  
Promising Alternative ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples ◽  
Endemic Areas ◽  
Collection Sites

Abstract Background: Schistosomiasis mansoni is a neglected tropical disease endemic in Brazil caused by Schistosoma mansoni, which is transmitted by Biomphalaria snails. Among all measures to control and eliminate the disease, accurate mapping and monitoring of snail breeding sites for susceptible and/or infected hosts in endemic areas are recommended. Parasitological methods are frequently used to identify infected snails, although they have many limitations, often providing false-negative results. Loop-mediated isothermal amplification (LAMP) is a promising alternative method for a more sensitive, rapid, and cost-effective diagnosis. However, standardization of LAMP assays is challenging due to the variety of parasites that are co-endemic with S. mansoni, and their varying prevalence rates in different areas. In this work, we aimed to optimize a LAMP assay for the detection of S. mansoni in Biomphalaria snails from endemic areas in the state of Minas Gerais, Brazil. Methods: A total of 1,001 snails were collected in five municipalities of the Mucuri and Jequitinhonha Valleys. Snails were pooled and squeezed according to the collection site to detect the presence of the larval forms of S. mansoni and other trematodes. Pooled snails were submitted to pepsin digestion and DNA extraction. Then molecular assays were performed for the species-specific identification and characterization of the samples. A LAMP assay was optimized and validated using laboratory and field samples. Results: Using the parasitological method, S. mansoni cercariae were detected in snails from two collection sites. Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites by PCR-RFLP. Multiplex PCR, LS-PCR, and conventional PCR allowed the detection of positive snails in four additional sites. The optimized LAMP assay was effective in detecting the presence of S. mansoni infection with 100% sensitivity, 91.66% specificity, and a Kappa index of 0.88, when compared to LS-PCR and conventional PCR. Conclusions: Our findings suggest that LAMP is a good alternative for the detection and monitoring of transmission foci of S. mansoni, as it enabled the detection of infection three times more than the parasitological examination and is more applicable directly in the field when compared to other molecular approaches.

A rapid, sensitive, specific and visual detection of Salmonella in retail meats using the loop-mediated isothermal amplification (LAMP), targeting the invA gene

2021 ◽  
Author(s):  
Can Wang ◽  
Ziheng Xu ◽  
Xuejiao Hou ◽  
Min Wang ◽  
Chenyu Zhou ◽  
...  
Keyword(s):  
Food Safety ◽  
Visual Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
National Standard ◽  
Lamp Method ◽  
Analytical Sensitivity ◽  
Conventional Pcr ◽  
Inva Gene ◽  
Loop Mediated Isothermal Amplification

Salmonella is one of the major pathogenic bacteria causing food-borne diseases. The rapid detection of Salmonella in food is of great significance to food safety. In this study, the loop-mediated isothermal amplification (LAMP) method was developed and the primers were designed targeting the invA gene of Salmonella. Then, the standard samples of recombinant invA-plasmid and 100 retail meat samples were tested by LAMP and compared with the results tested by the conventional PCR and the routine China National Food Safety Standard Methods for Food Microbiological Examination-Salmonella (GB/T4789.4-2016), respectively. The results showed that Salmonella strains of 8 different serotypes were amplified successfully by the developed LAMP assay. And, it was 1000-fold more sensitive than the conventional PCR with the analytical sensitivity of 8×102 copies/μL of the standard sample of invA-plasmid. The results were visualized directly by adding Calcein/MnCl2 into the LAMP reaction tube and the positively amplified products turned green after an incubation of 2 min. In the parallel detection, the positive rate of Salmonella by the LAMP assay was highly correlated with the routine China national standard method. The results of the study demonstrated that the developed LAMP assay is a simple, rapid, strongly specific, highly sensitive and visual detection method for Salmonella.

scholarly journals Isolation, molecular identification and antibiogram profiles of Escherichia coli and Salmonella spp. from diarrhoeic cattle reared in selected areas of Bangladesh

2017 ◽  
Vol 2 (4) ◽  
pp. 587-595
Author(s):  
M Sohidullah ◽  
Md Shahidur Rahman Khan ◽  
Md Shafiqul Islam ◽  
Md Mehedul Islam ◽  
Saifur Rahman ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Research Work ◽  
Clinical Signs ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Salmonella Spp ◽  
Isolation And Identification ◽  
E Coli ◽  
Field Samples ◽  
Cultural Media

The present research work was undertaken to find out the passive causes of occurrences of diarrhoea in terms of age, sex, season and location differences through isolation and identification of the E. coli and Salmonella spp. using cultural, biochemical and molecular from the field samples of the diarrhoeic cattle and to study the antibiogram profiles of the isolated bacterial species. Considering above purposes, a total of 57 rectal swab samples were collected from the diarrhoeic cattle of Mymensingh sadar, Trishal, Valuka, Natore sadar and Gomostapur, Chapai Nawabganj. Different types of cultural media like Nutrient agar, MacConkey`s (MC) agar, Eosin Methylene Blue (EMB) agar, Salmonella-Shigella (SS) agar, Xylose-Lysine-Deoxycholate (XLD) agar and Blood agar were used to isolate and to study the cultural properties of the E. coli and Salmonella spp. Finally Gram’s staining and different biochemical tests were performed to identify those two bacterial species. Out of 57 samples, 27 were positive for E. coli and 8 were positive for Salmonella spp. On the basis of information from cattle owners and clinical signs the prevalence of diarrhoea was recorded as 30.99% and the pvalue was calculated as 0.001 (p<0.01) which was noted as highly significant. The prevalence percentages of the E. coli and Salmonella spp. were differed depending on different epidemiological parameters like age, sex, season and location. Moreover, the molecular identifications were further confirmed by means of PCR assay using specific primers for E. coli and Salmonella spp. This was done targeting 16S rRNA gene where they were found to be positive showing amplification of 585 bp for E. coli and 574 bp for Salmonella spp. From the study of the antibiogram profiles, it was revealed that E. coli were susceptible to ciprofloxacin, gentamicin and norfloxacin but resistant to tetracycline, erythromycin, amoxicillin and streptomycin whereas Salmonella spp. were susceptible to ciprofloxacin, gentamicin, amoxicillin and streptomycin but resistant to azithromycin, tetracycline and erythromycin. The findings of this research work would certainly help to select the proper antibiotics against diarrhoea in cattle of Bangladesh and to overcome the multi-drug resistant problem of the bacteria.Asian J. Med. Biol. Res. December 2016, 2(4): 587-595

scholarly journals Isolation and identification of bacterial pathogens from cloacal swabs of turkeys and their antimicrobial sensitivity patterns

2018 ◽  
Vol 3 (4) ◽  
pp. 419-425
Author(s):  
Polash Chandra Roy ◽  
Md Khaled Hossain ◽  
Nazmi Ara Rumi ◽  
Md Shajedur Rahman ◽  
Md Shahin Mahmud ◽  
...  
Keyword(s):  
Bacterial Pathogens ◽  
Bacterial Species ◽  
Research Work ◽  
Cloacal Swab ◽  
Easy Access ◽  
Salmonella Spp ◽  
Isolation And Identification ◽  
E Coli ◽  
Antimicrobial Sensitivity ◽  
Pass Through

The present study was carried for the isolation, identification of bacterial pathogens from cloacal swabs of turkeys during the period from January-June, 2016. The entire research work was conducted in the Department of Microbiology, Faculty of Veterinary and Animal Science, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur. The study was performed with 48 cloacal swab samples. The cloacal swab samples were collected carefully from three different Turkey Farms randomly and transferred aseptically to the laboratory. On the basis of morphology, staining, cultural and biochemical characteristics it was found that among the isolates 25(52.08%) samples were positive E. coli, 10(20.83%) samples were positive for Salmonella spp., 9(18.76%) samples were positive for both E. coli and Salmonella spp. and 4(8.33%) samples shown no growth in subculture media. Antibiogram profiles indicate that E. coli isolated were 100% sensitive to Azithromycin, Kanamycin and Ciprofloxacin, 80% sensitive to Cefradine, Vancomycin and Levofloxacin, 60% sensitive to Cefotetan and Nitrofurantoin and 40% sensitive to Erythromycin. The isolates were 100% resistant to Cloxacillin and Cefixime. On the other hand, Salmonella spp. were 100% sensitive to Azithromycin, Kanamycin, Levofloxacin and Ciprofloxacin, 80% sensitive to Nitrofurantoin and Teicoplanin, 60% sensitive to Vancomycin, Erythromycin and Cefixime and 20% sensitive to Cefotetan. The isolates were 100% resistant to cefradine and cloxacillin. So, for E. coli Azithromycin, Kanamycin and Ciprofloxacin were more sensitive and for Salmonella spp. Azithromycin, Kanamycin, Ciprofloxacin and Levofloxacin were highly sensitive. Diversified bacterial species were present in cloacal swabs of Turkeys. However, E. coli, Salmonella spp. infection might make the birds vulnerable for easy access of infection. It could be concluded that E. coli and Salmonella spp. may pass through the feces to the environment. It causes a potential human health hazards and can cause illness.Asian J. Med. Biol. Res. December 2017, 3(4): 419-425

scholarly journals Isolation and identification of bacterial flora from respiratory tract of healthy horses

2016 ◽  
Vol 13 (2) ◽  
pp. 239-246
Author(s):  
ZB Muktha ◽  
SML Kabir ◽  
MT Rahman
Keyword(s):  
Staphylococcus Aureus ◽  
Respiratory Tract ◽  
Bacterial Species ◽  
Bacterial Flora ◽  
Antibiotic Sensitivity ◽  
Aerobic Condition ◽  
Diffusion Method ◽  
Bacterial Isolates ◽  
Isolation And Identification ◽  
E Coli

This study was carried out during the period of July to December, 2014 in order to isolate and characterize bacterial flora present in the respiratory tract of healthy horses in and around BAU campus. Eighteen apparently healthy horses were used for the study. Swab samples were collected from the nasal cavity. The bacteria was isolated ,identified and characterized by cultural (aerobic condition),staining, biochemical and PCR technique. Each of the samples collected yielded at least one bacterial species. A total of 27 bacteria were isolated from the selected animals. The majority of the isolates (15/27, 55.56%) were Gram-positive and the rest (12/27, 44.44%) were Gram-negative. Bacterial isolates were Staphylococcus aureus (83.33%) and E. coli (66.66%).The antimicrobial susceptibility pattern of bacterial isolates was investigated by disc diffusion method. The antibiotic sensitivity test of Staphylococcus aureus revealed that the isolates were highly sensitive to ciprofloxacin, moderately sensitive to gentamicin and resistance to amoxicilin, ampicilin and erythromycin. On the other hand, E. coli showed moderately sensitive to ciprofloxacin and gentamycin, mildly sensitive to erythromycin and resistance to amoxicilin and ampicilin. Detection of E. coli and S. aureus from the respiratory tract of healthy horses were not unexpected. Ciprofloxacin and gentamicin could be used for therapeutic purpose, if diseases occur by these organisms in horses.J. Bangladesh Agril. Univ. 13(2): 239-246, December 2015

scholarly journals Isolation and Identification of Bacterial Flora from Internal Organs of Broiler and Their Antibiogram Studies

2013 ◽  
Vol 1 (2) ◽  
pp. 72-75 ◽  
Author(s):  
Sampa Rani Roy ◽  
Md Bahanur Rahman ◽  
Jayedul Hassan ◽  
KHM Nazmul Hussain Nazir
Keyword(s):  
Bacterial Species ◽  
Bacterial Flora ◽  
Research Work ◽  
Easy Access ◽  
Sample Type ◽  
Salmonella Spp ◽  
Isolation And Identification ◽  
Internal Organs ◽  
E Coli ◽  
Bacillus Spp

The present research work was carried out for the isolation and identification of bacterial flora from internal organs of broiler during the period from January 2012 to June 2012. Ten Hubbard classic broiler bird were purchased from retail market in Mymensingh, Bangladesh. The birds were sacrificed and their liver, lung, esophagus, duodenum and tracheal swab samples were collected (n=50). Using standard bacteriological techniques, Escherichia coli was isolated from 26 (52%) samples. Similarly, Salmonella spp., Staphylococcus spp., Bacillus spp., and Pasteurella spp. were isolated from 15 (30%), 10 (20%), 9 (18%) and 4 (8%) samples, respectively. On the basis of individual sample type, E. coli could be isolated from 8 (80%) duodenum samples being the most prevalent organism. On the other hand, Salmonella spp., Staphylococci spp., Bacillus spp. and Pasteurella spp. were identified in 5 (50%) lungs, 5 (50%) liver, 4 (40%) duodenum and 2 (20%) lungs samples, respectively. Among these isolated bacteria, E. coli was found to be pathogenic for mice. Antibiogram studies revealed that Ciprofloxacin was highly sensitive against all the isolated bacteria. Diversified bacterial species are prevalent in broiler. However, E. coli and Salmonella spp. infection might make the bird vulnerable for easy access of infection. Proper vaccination and use of selective antibiotics are crucial in protecting broilers from these pathogens. DOI: http://dx.doi.org/10.3329/mh.v1i2.14094 Microbes and Health, 2012 1(2): 72-75

scholarly journals Isolation and Identification of Bacterial Agents Causing Clinical Mastitis in Cattle in Mymensingh and Their Antibiogram Profile

2014 ◽  
Vol 2 (1) ◽  
pp. 19-21
Author(s):  
Md Tanvir Rahman ◽  
Md Shafiqul Islam ◽  
Mahmudul Hasan
Keyword(s):  
Dairy Cattle ◽  
Bacterial Species ◽  
Clinical Mastitis ◽  
Clinical Form ◽  
Isolation And Identification ◽  
E Coli ◽  
The World ◽  
Bacillus Spp ◽  
Microbial Etiology ◽  
Staphylococcus Spp

Mastitis is a serious problem for the dairy cattle in many countries of the world including Bangladesh. Among the microbial etiology bacteria plays a major role in the onset of the clinical form of the disease. Many of these bacteria are resistant to one or more antibiotics thus make the mastitis cases difficult to cure. In the present study Staphylococcus spp., Streptococcus spp., Bacillus spp. and E. coli were identified as the dominant bacterial species causing clinical mastitis in cattle in Mymensingh. Antibiogram study revealed chloramphenicol and erythromycin as the most effective antibiotic for the treatment of mastitis in these animals except those caused by Bacillus spp. and E. coli, respectively. Microbes and Health, June 2013, 2(1): 19-21DOI: http://dx.doi.org/10.3329/mh.v2i1.17258

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