Online exercise for the design and simulation of PCR and PCR-RFLP experiments

Millán, Rosario María San; Martínez-Ballesteros, Ilargi; Rementerı́a, Aitor; Garaizar, Javier; Bikandi, Joseba

doi:10.1186/1756-0500-6-513

Cited by 60 publications

(44 citation statements)

References 10 publications

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“…Complementarity with the human genome was also verified (Altschul et al ). To confirm whether the primer pair actually works in silico , it was tested in the online PCR programme (http://insilico.ehu.es) (San Millán et al ).…”

Section: Methodsmentioning

confidence: 99%

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Machado¹,

Lyon

Rocha-Silva³

et al. 2020

J Appl Microbiol

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Aims Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae’s DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. Methods and Results The specific target region, RLEP, of M. leprae’s genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. Conclusions In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. Significance and Impact of the Study The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.

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Section: Methodsmentioning

confidence: 99%

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Machado¹,

Lyon

Rocha-Silva³

et al. 2020

J Appl Microbiol

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“…Pulse-field gel electrophoresis (PFGE) digestion and construction of the dendrogram was done in silico using the website http://insilico.ehu.es/digest/. 24,25 The enzyme used for the digestion was Xbal and lambda ladder was used to compare the bands.…”

Section: Methodsmentioning

confidence: 99%

Phylogenetic Analysis of Antibiotic Resistance Genes and Virulence Genes of Klebsiella species in silico

Nahar

Rashid

2017

Dhaka Univ. J. Pharm. Sci

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A total of twelve isolates were screened for virulence and antibiotic resistance genes associated with Klebsiella pneumoniae infections. Virulence and antibiotic resistance genes were detected by in silico PCR amplification. Iron uptake protein entB was detected in 66.67% (n=8) of the isolates while no isolate was found to harbour chelating agent irp2. Iron uptake system kfu, involved in purulent tissue infections and capsule formation, was identified in 25% (n=3) of the isolates. Regulator of mucoid phenotype A, rmpA was not found in any of the isolates. The wabG gene, responsible for urinary tract infections was found in seven K. pneumoniae strains. Five uge positive strains might play role in the pathogenicity of K. pneumoniae infections. About 83.33% of the isolates were positive for type 1 fimbriae fimH1 while no type 3 fimbriae mrkD gene was found. Complement reaction blocked by plasmid traT gene was not observed in Klebsiella species while eight isolates harboured outer membrane lipoprotein, ycfM which protects Klebsiella species from antibiotics. Antibiotic resistance genes bla TEM and bla SHV were detected in 33.33% (n=4) and 66.67% (n=8) of the isolates while 25% isolates carried both bla TEM and bla SHV genes. Genotype 1 carried fimH1 and ycfM genes while all the virulence genes studied were present in genotype 2 and 3. The bla SHV gene was detected in all the genotypes while bla TEM gene was found in only genotype 1 and 3. The findings of this study would be helpful to predict virulence gene associated with Klebsiella infections. This data also helps us to choose antibiotics for treating Klebsiella infections. By assessing the genotypic distribution of antibiotic resistance gene, correct antibiotic can be used to treat the infection. This could help reduce emergence of antibiotic resistance since it is known that incorrect choice of antibiotics contributes to antibiotic resistance.

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“…PFGE digestion: Pulse-field gel electrophoresis (PFGE) digestion and construction of the dendrogram was done in silico using the website http://insilico.ehu.es/digest/. 33,34 The enzyme used for the digestion was SgrDl and lambda ladder was used to compare the bands.…”

Section: Methodsmentioning

confidence: 99%

In silico Assessment of the Genotypic Distribution of Virulence and Antibiotic Resistance Genes in Vibrio cholerae

Nahar

Rashid

2017

Dhaka Univ. J. Pharm. Sci

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Vibrio cholerae has long been reported as an important cause of death in developing countries. The study detected the virulence and antibiotic resistance gene of eight V. cholerae isolates through in silico tools. Cholera toxins, ctxA and ctxB were found in six isolates (75%). Seventy-five percent isolates were also found to be positive for zonula occludens toxin, zot which is known to increase the permeability by altering the tight junction of the small intestine. Accessory cholera enterotoxin ace, responsible for fluid accumulation, was detected in four V. cholerae strains. Seven isolates (87.5%) were positive for toxin-coregulated pilus, tcp which helps the bacteria to adhere to gut mucosa. Both ompW and toxR genes were found in 87.5% of the isolates. Twenty-five percent isolates harboured strA, strB, sulII, dfrA1, floR genes and SXT element demonstrating that they were multidrug-resistant (MDG) bacterium. One isolate was found to be positive for tetA gene while no erythromycin resistance gene, ermA and ermB was found. Virulence genes were found in all genotypes indicating that their distribution was not genotypeoriented while genotype 2 harboured no antibiotic resistance genes. This data helps to predict virulence genes associated with cholera and also demonstrates the presence of antibiotic resistance genes. Bacteria acquired the antibiotic resistance gene through natural process which cannot be stopped. So by analyzing the resistance pattern we can choose appropriate antibiotics. In silico study helps us to predict the antibiotic resistant genotypes and can easily identify antibiotic resistant strains which help us to treat cholera infections and reduce the morbidity and mortality rate of the infected individuals.

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Online exercise for the design and simulation of PCR and PCR-RFLP experiments

Cited by 60 publications

References 10 publications

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Phylogenetic Analysis of Antibiotic Resistance Genes and Virulence Genes of Klebsiella species in silico

In silico Assessment of the Genotypic Distribution of Virulence and Antibiotic Resistance Genes in Vibrio cholerae

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