Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

Accurate analysis of DNA sequence variation in not only humans and animals but also other organisms has played a significant role in expanding our knowledge about genetic variety and diversity in a number of different biological areas. The search for an understanding of the causes of genetic variants and mutations has resulted in the development of a simple laboratory technique, known as the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, for the detection of single nucleotide polymorphisms (SNPs). PCR-RFLP allows rapid detection of point mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by gel electrophoresis after staining with ethidium bromide (EtBr). This convenient and simple method is inexpensive and accurate for SNP genotyping and especially useful in small basic research studies of complex genetic diseases. The whole protocol takes only a day to carry out.     

Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism

Abstract. Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty-seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (R). Five restriction fragment length patterns were polymorphic in C. hominivorax while all fragment patterns were fixed in C. macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than 2.50.     

Discrimination between Anguilla anguilla and A. rostrata by polymerase chain reaction-restriction fragment length polymorphism analysis

HinfI polymorphism, detected in the mitochondrial DNA, discriminates between the Atlantic eel species Anguilla anguilla and A. rostrata .     

Detection and investigation of Campylobacter jejuni by polymerase chain reaction-restriction fragment length polymorphism analysis

A molecular typing approach for Campylobacter jejuni with restriction fragment length polymorphism (RFLP) analysis of the flagellin gene flaA in C. jejuni, was generated and studied. Using polymerase chain reaction (PCR)-RFLP with the restriction endonuclease Mbo I, it was demonstrated that C. jejuni could be divided into four types. Genotypic analysis of C. jejuni by PCR-RFLP is a valuable technique for epidemiological typing.     

Typing of food-borne Listeria monocytogenes by polymerase chain reaction-restriction enzyme analysis and amplified fragment length polymorphism

The applicability of polymerase chain reaction-restriction enzyme analysis (PCR-REA) and amplified fragment length polymorphism (AFLP) for typing of food borne Listeria monocytogenes strains was tested. A panel of 43 L. monocytogenes strains isolated from food, mostly serovars 1/2b or 1/2a, were analysed by the optimized PCR-REA oriented to inlA and inlB genes and by AFLP. By PCR-REA, five types of profiles were obtained. By AFLP, the strains were separated into 11 types and 18 subtypes forming two major clusters. PCR REA was a relatively straightforward method for typing food-borne L. monocytogenes with a moderate discrimination power. AFLP was a more complex but a highly discriminative and reproducible method.     

Taenia saginata: differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different DraI-RFLP pattern: a two-band pattern (421 and 100 bp) for T. saginata and a three-band pattern (234, 188, and 99 bp) for T. solium was observed allowing the two species to be separated. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T. saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). Of these, three showed a T. solium pattern and five a T. saginata pattern.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.     

Differentiation of sheeppox and goatpox viruses by polymerase Chain reaction-restriction fragment length polymorphism

In the present study, the partial gene sequences of P32 protein, an immunogenic envelope protein of Capripoxviruses (CaPV), were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates, and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains. A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene. The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels. Phylogenetic analysis showed three distinct clusters of SPPV, GTPV and Lumpy skin disease virus (LSDV) isolates. Further, multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I, which is present in SPPV isolates studied. This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains. The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV. The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.     

Species identification in seven small millet species using polymerase chain reaction-restriction fragment length polymorphism of trnS-psbC gene region.

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.     

Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis

Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results.     

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.     

Utility of MtCOI polymerase chain reaction-restriction fragment length polymorphism in differentiating between Q and B whitefly Bemisia tabaci biotypes

The invasive, insecticide-resistant, Q whitefly biotype, has gradually spread to other countries including the US via human-mediated movement of plant materials. We assessed the utility of the VspI -based mtCOI (mitochondrion cytochrome oxidase I) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique as a rapid, cost-effective, and reliable alternative for differentiating the Q from the dominant B biotype in Arizona. Using the standard mtCOI gene sequencing and mtCOI PCR-RFLP techniques, we biotyped eight whitefly strains of five individuals each collected from poinsettia and cotton at different locations in Arizona. Complete concordance was observed between the two methods, with three strains being identified as the Q biotype and five samples as the B biotype. We also scanned the mtCOI gene sequences for VspI polymorphisms in the B and Q biotype whiteflies currently available in the GenBank database. This global screening revealed the existence of three and four VspI polymorphic types for the Q and B biotypes, respectively. Nevertheless, all three VspI polymorphic Q biotype whiteflies shared a common and unique VspI site that can be used to differentiate Q biotype from the four VspI polymorphic B biotype whiteflies identified. These results demonstrate that the VspI -based mtCOI gene PCR-RFLP provides a reliable diagnostic tool for differentiating the Q and B biotype whiteflies in the US and elsewhere.     

Rapid polymerase chain reaction-restriction fragment length polymorphism method for discrimination of the two Atlantic cryptic deep-sea species of scabbardfish

The present investigation provides an efficient diagnostic method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis to discriminate between two cryptic species of scabbardfish, Aphanopus carbo and A. intermedius, with commercial relevance in several European fish markets. Two DNA fragments from the mtDNA, including control region and partial cytochrome oxidase subunit I genes of about 1100 bp and 700 bp, respectively, were isolated by PCR amplification. Digestion of the amplicon including the control region with HaeII and the amplicon including the COI gene with Sau3AI restriction enzymes allowed an unequivocal discrimination between the two scabbardfish species. This PCR–RFLP method allowed a clear and rapid discrimination of the trichiurid species studied.     

Polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial rRNA genes from yellowtail rockfish

A 1600 base pair region of the mitochondrial rRNA genes from 74 yellowtail rockfish, collected from three north-east Pacific localities, was amplified via PCR. RFLP analysis yielded a total of 33 restriction sites allowing examination of 139 base pairs or 0·8% of the mtDNA molecule. Essentially no variation was detected within or among the three populations.     

Use of mitochondrial cytochrome oxidase I polymerase chain reaction-restriction fragment length polymorphism for identifying subclades of Bemisia tabaci Mediterranean group

The Mediterranean group (commonly known as Q biotype; hereafter MED) of the sweetpotato whitefly, Bemisia tabaci (Gennadius), originated in the Mediterranean region, but it now has been found in at least 10 countries outside the Mediterranean. Collections of B. tabaci from some of these countries exhibit different pest behaviors and pesticide resistance characteristics, yet all may be classified as MED. A phylogenetic analysis of 120 mitochondrial cytochrome oxidase I (mtCOI) sequences (JN966761-JN966880) of MED whiteflies collected in Arizona and of 417 retrieved from the GenBank database resolves the MED into five subclades, designated as Q1-Q5. Only subclades Q1 and Q2 have been detected in the United States. Q1 and the other four subclades (Q2-Q5) differ in the number or position of the AluI recognition sites. Based on the differences in the AluI recognition sites reported here and the previously reported differences in VspI recognition sites, we developed a simple diagnostic technique to identify subclades Q1-Q5 by using mtCOI polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP). A test of a worldwide collection of whiteflies demonstrates that this combination mtCOIPCR-RFLP technique can reliably distinguish not only the MED from the Middle East-Asia Minor 1 group but also the Q1 from any of the other four MED subclades.     

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.     

Morphological and polymerase chain reaction-restriction fragment lenght polymorphism characterization of Biomphalaria kuhniana and Biomphalaria amazonica from Colombia

In Colombia, five Biomphalaria planorbid species are known: B. kuhniana, B. straminea, B. peregrina, B. canonica and B. oligoza(var. B. philippiana). Among them, B. straminea is intermediate host of Schistosoma mansoni and B. peregrina has been found to be experimentally susceptible to this parasite. B. straminea is commonly confused with B. kuhniana and they have been clustered together with B. intermedia in the complex named B. straminea. The difficulties involved in the specific identification, based on morphological data, have motivated the use of new techniques as auxiliary tools in cases of inconclusive morphological identification of such planorbid. In the present study, five Biomphalaria populations from the Colombian Amazon region and from Interandian Valleys were morphologically identified and characterized by polymerase chain reaction-restriction fragment lenght polymorphism directed at the internal transcribed spacer region of the rRNA gene, followed by digestion of the generated fragment with restriction enzymes (DdeI, AluI, RsaI, MvaI and HaeIII). Known profiles of the Brazilian species B. straminea, B. peregrina, B. kuhniana, B. intermedia and B. amazonica, besides B. kuhniana from Colombia, were used for comparison. The five populations under study were morphologically and molecularly identified as B. kuhniana and B. amazonica.     

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.     

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and plasmid profile of soil and clinical isolates of Nocardia.

The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for generic and species-specific differentiation of Nocardia from other morphologically similar bacterial pathogens. To examine the utility of the PCR-RFLP approach in species identification, genomic DNA was prepared from 40 soil isolates, 10 clinical isolates and 8 reference strains of Nocardia. A set of oligonucleotide primers was designed from the consensus sequence of the highly conserved groEL gene that encodes the 65-kDa heat shock protein (hsp 65). The primers selectively amplified 422 bp DNA from the genomic DNA of all Nocardia species and isolates. The digestion of the amplicons with the restriction enzyme MspI produced DNA fragments that could differentiate between different Nocardia species regardless of their origin. Additionally, the RFLP patterns obtained with restriction enzymes MspI and BsaHI resulted in the differentiation of six Nocardia species which were earlier identified by biochemical tests. Apart from soil isolates of N. asteroides, which had shown some degree of genotypic polymorphism with BsaHI, the remaining taxa yielded more consistent results. Our results on the isolation of plasmids indicated that their occurrence is not a consistent feature in Nocardia species. It is neither related to the source of origin (clinical versus saprobic), nor to virulence, anti-microbial resistance or species specificity.