• Journal of fish pathology
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• v.21 no.3
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• pp.181-188
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• 2008

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect and identify four fish viruses, fish iridovirus, viral hemorrhagic septicaemia virus (VHSV), viral nervous necrosis virus (VNNV), hirame rhabdovirus (HRV). Four viruses were detected by PCR with each specific primers. Identification of iridovirus was achieved by digesting the PCR amplified fragment with a restriction enzyme ApaⅠ. It was possible to distinguish positive from false positive PCR amplicons of VHSV by RFLP of PstⅠ or HindⅢ restriction enzymes. VNNV was identified using RFLP of BamHⅠrestriction enzyme and HRV was identified by XbaⅠ restriction enzyme. This approach can be used for more rapid, simple and specific diagnosis of fish viral diseases.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.2
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• pp.262-266
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• 2017

Objective: This study was conducted to screen scrotal hernia in domesticated swine from selected breeders in the Philippines. This defect is associated with a cytosine to thymine mutation in the BCL-2 associated X protein (BAX) gene of swine. Methods: Genetic screening was done by DNA extraction followed by amplification and digestion using polymerase chain reaction-restriction fragment length polymorphism, amplifying the 416 bp region of the BAX gene that was subjected to digestion using the Ear I enzyme. Sequencing was also conducted to validate the results. Results: Results revealed that out of 538 samples tested, 411 (76.4%) of the samples were found to be normal whereas the remaining were carriers of the mutation in which 80 (14.9%) were heterozygous mutants and 47 (8.7%) were homozygous mutants. Pietrain breed was found to have the highest incidence. Conclusion: Having a scrotal hernia eliminates the chances of using the boar as a breeder stock because the following generations arising from it would most likely exhibit herniation. It is therefore advised to establish a genetic screening method for Scrotal Hernia in the Philippines to eliminate the negative gene from the herd.

• Journal of Food Hygiene and Safety
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• v.23 no.4
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• pp.314-318
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• 2008

This research aimed to compare the detection methods of Anisakis simplex in Sea fish by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and macroscopic inspection. We examined 18 Trichiurus lepturus, 11 Scomber japonicus, and 65 Todarodes pacificus collected from the retail markets in the areas of Uljin, Kyuonggi province and Seoul. As the result of examinations, we found that detection rate of Anisakis simplex by macroscopic observation was 89% in Trichiurus lepturus, 90.9% in Scomber japonicus, 32.3% in Todarodes pacificus. The detection rate of Anisakis simplex by PCR-RFLP was 77.7% in Trichiurus lepturus, 81.8% in Scomber japonicus, 26.1% in Todarodes pacificus. We could conclude that PCR-RFLP method of Anisakis simplex was more specific rather than macroscopic observation.

• Fisheries and Aquatic Sciences
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• v.11 no.3
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• pp.133-139
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• 2008

Molecular techniques, including restriction fragment length polymorphism(RFLP) and polymerase chain reaction-single strand conformational polymorph isms(PCR-SSCP), were developed to identify salted, dried threadfin(Eleutheronema tetradactylum) and white herring(Ilisha elongata) fish. Using PCR with universal primers, conserved 367-bp fragments of the cytochrome b gene were amplified from fresh fish samples and sequenced. The sequences were then searched for specific restriction sites. The digestion of the PCR products with the endonucleases AvaI, FokI, MboII, and MspI generated RFLP, which was used to identify the commercial products. Similarly, the amplified PCR-SSCP products were developed and the products tested. Overall, similar patterns were found in the majority of the fresh and processed products. Based on the results, both RFLP and PCR-SSCP were useful in determining and validating the authenticity of the fish species used to prepare the commercial salted, dried products. A similar approach can be applied to other species.

• Journal of Veterinary Clinics
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• v.27 no.1
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• pp.23-28
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• 2010

Mycobacterium avium ssp paratuberculosis, intracellular bacteria that can cause chronic granulomatous enteritis in cattle, continues to pose significant economic losses and health problem with high prevalence. The purpose of this study is the polymerase chain reaction (PCR)-base strategy for early detection of M. avium ssp paratuberculosis in whole blood. Blood samples were collected from korean cattles in Jeonbuk, Korea. The 16 out of 88 serum samples were detected M. partuberculosis by ELISA. Then samples of infected 8 Korean cattles were amplified by PCR. The PCR amplified targets are 16s rDNA and heat shock protein 65kDa (hsp 65). The 16s rDNA provided a highly sensitive and specific tool for the direct detection of mycobacteria. In addition M. avium was confirmed characteristically by the hsp65. Finally there were sure to M. avium ssp paratuberculosis by IS900 PCR. The restriction fragment length polymorphism was identified by PCR amplifications and subsequence restriction enzyme digestions with Pst I of a hsp65. These results indicate that confirm M. avium with 16s rDNA, hsp65 and a restriction fragment length polymorphism in the hsp65 gene can be seem the other pattern. Therefore, these results can be used for clinical direct detections of M. avium ssp paratuberculosis in whole blood of Korean cattle and also to be used epidemiological researches.

• Journal of Periodontal and Implant Science
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• v.29 no.2
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• pp.349-355
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• 1999

The present study has been performed to develop a PCR technology to identify human immunoglobulin(Ig) allotypes with restriction fragment length polymorphism(RFLP) using a probe. Genomic DNA were ampilified with PCR tecnology using primers from peripheral blood lymphocytes of 10 periodontal patiens, whose Ig allotypes have been pre-determined by serological tecnique using heagglutination technique. The result indicated that the RFLP patterns could successfully differentiate the Ig allotypes, which suggests that this technology can be developed as a tool useful for population genetics studies.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.10
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• pp.1487-1492
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• 2014

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.12
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• pp.1691-1695
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• 2005

The bovine lymphocyte antigen (BoLA)-DRB3 gene encodes cell surface glycoproteins that initiate immune responses by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. Since different alleles favour the binding of different peptides, DRB3 has been extensively evaluated as a candidate marker for associations with various bovine diseases and immunological traits. For that reason, the genetic diversity of the bovine class II DRB3 locus was investigated by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). This study describes genetic variability in the BoLA-DRB3 in Iranian Golpayegani Cattle. Iranian Golpayegani Cows (n = 50) were genotyped for bovine lymphocyte antigen (BoLA)-DRB3.2 allele by polymerase chain reaction and restriction fragment length polymorphism method. Bovine DNA was isolated from aliquots of whole blood. A two-step polymerase chain reaction followed by digestion with restriction endonucleases RsaI, HaeIII and BstYI was conducted on the DNA from Iranian Golpayegani Cattle. In the Iranian Golpayegani herd studied, we identified 19 alleles.DRB3.2${\times}$16 had the highest allelic frequency (14%), followed by DRB3.2${\times}$7 (11%). Six alleles (DRB3.2${\times}$25, ${\times}$24, ${\times}$22, ${\times}$20, ${\times}$15, ${\times}$3) had frequencies = 2%. Although additional studies are required to confirm the present findings, our results indicate that exon 2 of the BoLA-DRB3 gene is highly polymorphic in Iranian Golpayegani Cattle.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.2
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• pp.83-89
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• 2015

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV (human immuno deficiency virus) infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. M. tuberculosis was detected by two-tube nested polymerase chain reaction (PCR) and non-tuberculous mycobacteria was detected by PCR-restriction fragment length polymorphism (RFLP) with Msp I. Result of niacin test is equal to result of two-tube nested PCR after culture for M. tuberculosis. In this study, acid fast bacilli stain (AFB. stain) >2+ case, Detection of Mycobacteria is similar to result before culture and after culture. AFB. stain <1+ case, result of mycobacteria is distinguished. Conclusionly, these results suggest that identification of mycobacteria must go side by side both culture and PCR for more fast and accuracy.

• Journal of Korean Society on Water Environment
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• v.22 no.4
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• pp.590-598
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• 2006

In-situ Air sparging (IAS) is a groundwater remediation technique, in which organic contaminants volatilize into air form the saturated to vadose zone. This study was carried out to evaluate the effect of sludge and soil microbial community structure on air sparging of Total Petroleum Hydrocarbons (TPH) contaminated groundwater soils. In the laboratory, diesel (10,000 mg TPH/kg) contaminated saturated soil. The Air was injected in intermittent (Q=1500 mL/min, 10 minute injection and 10 minute idle) modes. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for experiment with sludge soil samples that were closely related to Agrococcus, Flavobacterium, Thermoanaerobacter, Flexibacter and Shewanella, etc, in the clone library. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil the fate of microorganisms in natural microbial community.