New Set Of Primers Research Articles

Widespread use of real-time PCR for rickettsial diagnosis: Figure 1

We report 2years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R.conorii, five Rickettsia sibirica mongolitimonae, four R.slovaca, two R.australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.

An improved polymerase chain reaction method for genetic testing of spinocerebellar ataxia type 3

The development of a reliable PCR assay for genetic testing of spinocerebellar ataxia type 3. Touchdown PCR conditions were tested and different primer sets were evaluated with genomic DNA from blood sample of patients suffering from spinocerebellar ataxia type 3 (SCA3). An improved PCR assay was developed with a new set of primers and using the optimized touchdown PCR protocol. This new assay had been successfully employed in the screening of one identificated SCA3 family. Results from the present study document a simple and reliable PCR assay for genetic testing of SCA3. Strategies used in the present study may find applications in the optimization of PCR assay for triplet expansion with GC rich in the sequence context.

Gene Cloning and Homology Modeling of the 3-Oxoacyl-ACP Synthase from Aeromonas hydrophila for Drug Discovery

Fatty acid biosynthesis in the Aeromonas hydrophila plays an important role in the formation of cell membrane and cell viability. The 3-oxoacyl- acyl carrier protein (ACP) synthase fadH and fadB from A. hydrophila have contributed in the initiation and elongation of fatty acid biosynthesis. In this study, we have designed new set of primers for amplification of the open reading frame of fadH and fadB. Cloning and sequencing of these amplified genes, analyzed that the G+C content of fadH and fadB were 63.3% and 68.79% respectively. The homology modeling was performed to generate the 3- D structure of fadH and fadB; and showed that more than 90% amino acid residues in allowed region of Ramachandranan plot. These findings provide a new avenue for better understanding, the role of genes encoding proteins involved in the fatty acid biosynthesis and thereby using as a potential and novel target for structure based drug designing to control the infections of A. hydrophila.

Molecular detection and cloning of thermostable hemolysin gene from Aeromonas hydrophila

Aeromonas hydrophila is a major bacterial pathogen associated with hemorrhagic septicemia in aquatic and terrestrial animals including humans. There is an urgent need to develop molecular and immunological assays for rapid, specific and sensitive diagnosis. A new set of primers has been designed for detection of thermostable hemolysin (TH) gene (645 bp) from A. hydrophila, and sensitivity limit for detection of TH gene was 5 pg. The TH gene was cloned, sequenced and analyzed. The G+C content was 68.06%; and phylogeny was constructed using TH protein sequences which had significant homology with those for thermostable and other hemolysins present in several bacterial pathogens. In addition, we have predicted the four and eight T-cell epitopes for MHC class I and II alleles, respectively. These results provide new insight for TH protein containing antigenic epitopes that can be used in immunoassays and also designing of thermostable vaccines.

A set of 100 chloroplast DNA primer pairs to study population genetics and phylogeny in monocotyledons.

Chloroplast DNA sequences are of great interest for population genetics and phylogenetic studies. However, only a small set of markers are commonly used. Most of them have been designed for amplification in a large range of Angiosperms and are located in the Large Single Copy (LSC). Here we developed a new set of 100 primer pairs optimized for amplification in Monocotyledons. Primer pairs amplify coding (exon) and non-coding regions (intron and intergenic spacer). They span the different chloroplast regions: 72 are located in the LSC, 13 in the Small Single Copy (SSC) and 15 in the Inverted Repeat region (IR). Amplification and sequencing were tested in 13 species of Monocotyledons: Dioscorea abyssinica, D. praehensilis, D. rotundata, D. dumetorum, D. bulbifera, Trichopus sempervirens (Dioscoreaceae), Phoenix canariensis, P. dactylifera, Astrocaryum scopatum, A. murumuru, Ceroxylon echinulatum (Arecaceae), Digitaria excilis and Pennisetum glaucum (Poaceae). The diversity found in Dioscorea, Digitaria and Pennisetum mainly corresponded to Single Nucleotide Polymorphism (SNP) while the diversity found in Arecaceae also comprises Variable Number Tandem Repeat (VNTR). We observed that the most variable loci (rps15-ycf1, rpl32-ccsA, ndhF-rpl32, ndhG-ndhI and ccsA) are located in the SSC. Through the analysis of the genetic structure of a wild-cultivated species complex in Dioscorea, we demonstrated that this new set of primers is of great interest for population genetics and we anticipate that it will also be useful for phylogeny and bar-coding studies.

A specific polymerase chain reaction test for the identification of Streptococcus pneumoniae

Using an approach based on the comparison of arbitrary primer polymerase chain reaction (PCR) genomic profiles from oral streptococci and Streptococcus pneumoniae strains, we identified a 434-bp genomic fragment apparently specific for S. pneumoniae. From the nucleotidic sequence of this common fragment, a pair of primers was designed and tested on a set of strains comprising the major Streptococcus species. One species, S. anginosus, gave an amplification product of the same length as S. pneumoniae. Sequence comparison of the S. anginosus and S. pneumoniae amplicons revealed several variations which were used to define a new set of primers giving a 181-bp S. pneumoniae–specific fragment. The amplified fragment contains the 5′ terminal part of a gene encoding a putative sugar-specific permease and an intergenic sequence. The PCR test was evaluated on 257 strains of invasive S. pneumoniae corresponding to clinical isolates and on 153 non-pneumoniae oral streptococci strains; in addition, 3 S. pseudopneumoniae strains were tested. With these primers, an amplification product was only obtained with the S. pneumoniae strains. Moreover, the test was successfully evaluated on 10 atypical S. pneumoniae strains related to pneumococcal diseases. In this study, we therefore established the capacity of a simple PCR test to discriminate S. pneumoniae from other Streptococci (including S. pseudopneumoniae), thus allowing rapid and accurate diagnosis.

Detecting mutations in PfCRT and PfMDR1 genes among Plasmodium falciparum isolates from Saudi Arabia by pyrosequencing

The emergence of chloroquine resistance in Plasmodium falciparum is a significant public health problem where malaria is endemic. We aimed to evaluate the efficacy of pyrosequencing to assess chloroquine resistance among P. falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 (PfMDR1) genes, respectively. Blood samples (n = 121) from microscopically positive P. falciparum cases were collected. DNA was extracted, and fragments from each of the genes were amplified by PCR using new sets of primers. The amplicons were sequenced using a pyrosequencer. All of the 121 samples were amplified for assessment of the PfCRT K76T and PfMDR1 N86Y mutations. All of the samples amplified for the PfCRT 76T mutation harbored the ACA codon (121/121; 100%), indicating the presence of the 76T mutation. For the PfMDR1 N86Y mutation, 72/121 samples (59.5%) had the sequence AAT at that position, indicating the presence of the wild-type allele (86N). However, 49/121 samples (40.5%) had a TAT codon, indicating the mutant allele (Y) at position 86. This study shows that pyrosequencing could be useful as a high throughput, rapid, and sensitive assay for the detection of specific single nucleotide polymorphisms in drug-resistant P. falciparum strains. This will help health authorities in malaria-endemic regions to adopt new malaria control strategies that will be applicable for diagnostic and drug resistance assays for malaria and other life-threatening pathogens that are endemic in their respective countries.

An Updated Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of H5N1 Avian Influenza Viruses.

We designed a new set of primers for reverse transcriptase loop-mediated isothermal amplification (RTLAMP) to specifically amplify the HA gene of avian influenza viruses subtype H5N1. By testing nine H5N1 virus strains and 41 clinical samples collected in Northern Vietnam, we found that the new primers showed higher sensitivity and specificity than the previously published RT-LAMP primers and were comparable to the RT-PCR method currently recommended by WHO. These results suggest that our RT-LAMP assay may be a better choice as a diagnostic tool for current H5N1 influenza virus infection.

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

SUMMARYThe usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.

Development of Allele-Specific Single-Nucleotide Polymorphism-Based Polymerase Chain Reaction Markers in Cytochrome Oxidase I for the Differentiation of Bactrocera papayae and Bactrocera carambolae (Diptera: Tephritidae)

Differentiation of Bactrocera papayae Drew & Hancock and Bactrocera carambolae Drew & Hancock (Diptera: Tephritidae) based on morphological characters has often been problematical. We describe here a single-nucleotide polymorphism (SNP)-based polymerase chain reaction (PCR) assay to differentiate between these two species. For detection of SNPs, fragments derived from each species were amplified using two primer pairs, COIF/COIR and UEA7/UEA10, sequenced, and aligned to obtain a contiguous 1,517-bp segment. Two new sets of primers were designed based on the 11 SNPs identified in the region. Results of the SNP-PCR test using any one of these species-specific primer sets indicate that these two species could be differentiated on basis of presence or absence of a band in the gel profile. We also tested the SNP-PCR primers on Bactrocera umbrosa F., Bactrocera cucurbitae Coquillett, Bactrocera latifrons Hendel, and Bactrocera tau (Walker) but did not detect any band in the gel, indicating the likelihood of a false positive for B. papayae is nil. This SNP-PCR method is efficient and useful, especially for immature life stages or when only adult body parts of the two species are available for identification, as encountered often in quarantine work.

Multiple myeloma patients at peripheral blood stem cell harvest: Restricted usage of TCR beta variable families

The immune systems of multiple myeloma patients are suppressed by the disease itself, and this immunosuppression is further enhanced by standard therapies. The aim of our study was to evaluate the effects of initial chemotherapy and a peripheral blood mobilisation regimen on T-cell population diversity. Reverse transcription-polymerase chain reaction (RT-PCR) with a new set of primers, in combination with capillary electrophoresis, was established. The methodology was used to analyse the relative expression of 27 T-cell receptor beta variable gene families (BV families) in multiple myeloma patients undergoing peripheral blood stem cell harvest. We found that the overall BV family usage in these patients was restricted; the relative expression of 10 BV families was significantly depressed in patients compared to healthy donors. These findings demonstrate that the preparative regimen for autologous stem cell transplantation affects the T-cell population in terms of the restriction of its T-cell receptor diversity.

Universal primers for rapid detection of hytrosaviruses

Hytrosaviridae is a proposed virus family encompassing viruses that cause salivary gland hypertrophy (SGH) syndrome in infected insects and reduce the fertility in their dipteran insect hosts. They contain a large, double stranded DNA genome of 120–190 kbp. To date, these viruses have been detected only in adult Diptera. These include hytrosaviruses detected in various tsetse fly species ( Glossina spp.), the narcissus bulb fly Merodon equestris and the house fly Musca domestica. The limited number of hytrosaviruses reported to date may be a reflection of the frequent absence of external symptoms in infected adult flies and the fact that the virus does not cause rapid mortality. Based on the complete genome sequence of Glossinia pallidipes (GpSGHV) and Musca domestica (MdSGHV) salivary gland hypertrophy viruses, a PCR based methodology was developed to detect the viruses in these species. To be able to detect hytrosaviruses in other Diptera, five degenerate primer pairs were designed and tested on GpSGHV and MdSGHV DNA using gradient PCR with annealing temperatures from 37 to 61 °C. Two pairs of primers were selected from p74, two pairs from PIF-1 and one pair from ODV-e66 homologous proteins. Four primer pairs generated a virus specific PCR product on both MdSGHV and GpSGHV at all tested annealing temperatures, while the ODV-e66 based primers did not generate a virus specific product with annealing temperatures higher that 47 °C. No non-specific PCR product was found when using genomic DNA of infected flies as template DNA. These results offer new sets of primers that could be used to detect hytrosaviruses in other insects.

Specific PCR primers for the detection of isolates of Aspergillus carbonarius producing ochratoxin A on grapevine

Aspergillus carbonarius is the main fungus responsible for ochratoxin A (OTA) production on grapes and in wine. Fungal polyketide synthases are involved in OTA biosynthesis in Aspergillus species. The ketosynthase (KS) domain of an A. carbonarius gene encoding polyketide synthase (pks) was isolated, cloned and sequenced. The nucleotide sequence showed high similarity to KS domains isolated from other Aspergillus species. The sequence was used to design a new set of primers in order to identify potential producers of OTA from among isolates of A. carbonarius. The primers amplified all the A. carbonarius strains tested specifically, and did not amplify any other species of Aspergillus or Penicillium normally found on grapes or involved in OTA biosynthesis. Further, gene expression was related to OTA production. Both gene transcription and levels of the mycotoxin were higher when A. carbonarius was grown on YES medium at 15°C and 30°C, whereas a low transcription and mycotoxin presence were observed when the fungus was grown on PDB medium at the same temperatures. No transcription or OTA production were observed when the fungus was grown on either YES or PDB at 10°C. The primers will be useful for the detection of ochratoxigenic strains of A. carbonarius both in vineyards and during wine production.

Combined eukaryotic and bacterial community fingerprinting of natural freshwater biofilms using automated ribosomal intergenic spacer analysis

Biofilms are complex communities playing an important role in aquatic ecosystems. Automated ribosomal intergenic spacer analysis (ARISA) has been used successfully to explore biofilm bacterial diversity. However, a gap remains to be filled as regards its application to biofilm eukaryotic populations. The aim of this study is to use ARISA to detect eukaryotic population shifts in biofilm. We designed a new set of primers to focus specifically on the ITS1-5.8S-ITS2 region of diatoms and tested it on natural biofilms. Additionally, we tested universal primers, used previously to perform ARISA on fungal communities. Cloning and sequencing showed that the universal primer set amplified various eukaryotes, whereas the new set was diatom specific. The new set amplified a wider variety of diatoms. Therefore, the universal set is appropriate to study the general eukaryotic population shifts in biofilms, whereas the new set is more appropriate to study diatoms specifically. We used both primer sets, along with a bacterial set, to study the population shifts in natural river biofilms. Principal component analysis of the ARISA fingerprints revealed seasonal shifts that did not coincide for bacterial and eukaryotic communities. Therefore, the use of both eukaryotic and bacterial primers provides a useful insight to assess microbial succession in biofilms.

Comprehensive Detection of Phototrophic Sulfur Bacteria Using PCR Primers That Target Reverse Dissimilatory Sulfite Reductase Gene

A new set of primers for the detection of phototrophic sulfur bacteria in natural environments is described. The primers target the α-subunit of the reverse dissimilatory sulfite reductase gene (dsrA). PCR-amplification resulted in products of the expected size from all the phototrophic strains tested, including purple sulfur and green sulfur bacteria. Seventy-nine clones obtained from environmental DNA using the primers were sequenced and all found to be closely related to the dsrA of purple sulfur bacteria and green sulfur bacteria. This newly developed PCR assay targeting dsrA is rapid and simple for the detection of phototrophic sulfur bacteria in situ and superior to the use of culture-dependent techniques.

Diagnosis of Sugarcane yellow leaf virus in asymptomatic sugarcane by RT-PCR

Sugarcane yellow leaf virus (SCYLV) that causes yellow leaf disease (YLD) in sugarcane belongs to Polerovirus. The disease expression is commonly observed during 6 to 8 months stages of the crop and there is a need to diagnose the disease before the symptom appearance. We have standardized RT-PCR technique with a new set of primers to detect SCYLV in sugarcane in pre-symptomatic stages of YLD. During presymptomatic stage 33 of the 44 varieties studied tested positive to SCYLV in RT-PCR. During eighth month stage all the 44 varieties have shown characteristic disease symptoms and except one all of them tested positive to the virus in RT-PCR. The two RT-PCR assays performed separately during pre- and post-symptom expression stages in 44 varieties clearly revealed that most of the varieties have detectable level of the virus in asymptomatic stage. Expression of disease symptoms and presence of the virus in all the varieties except one very clearly indicated the severe virus infection in the tested varieties. The studies also proved the diagnostic efficiency of the new set of primers to detect SCYLV in asymptomatic plants.

First Report of a Natural Infection by Mexican Papita Viroid and Tomato Chlorotic Dwarf Viroid on Greenhouse Tomatoes in Mexico

In early 2008, tomato plants (Solanum lycopersicum) grown in a large greenhouse facility located near Mexico City exhibited general stunting, leaf chlorosis at the top of the diseased plant that later turned bronze or purple, and reduced-sized fruits. Initially, diseased plants were confined to a 5-ha greenhouse, but the disease quickly spread to two additional 5-ha greenhouses in the summer of 2008. By the end of 2008, approximately 5% of tomato plants in 35-ha of greenhouse were infected. Sixteen diseased samples were collected, twelve in 2008 and four in 2009. Bioassays through mechanical inoculation with leaf extracts of diseased samples demonstrated the transmissibility of the causal agent to plants of tomato cvs. Horizon or Rutgers, which expressed symptoms that were similar to those on the source plants. Serological or PCR assays were negative for several commonly occurring greenhouse tomato viruses. However, an expected size product (~196 bp) was consistently detected by reverse transcription (RT)-PCR using pospiviroid-specific primers Pospil-RE and Pospil-FW (4) in all symptomatic samples or from the mechanically inoculated tomato plants. Preliminary analysis with sequences obtained from direct sequencing of amplicons revealed one dominant sequence with 94% identity to Mexican papita viroid (MPVd) (GenBank Accessions Nos. L78454 and L78456-L78463). However, further analysis of the cloned cDNAs indicated a mixed infection of two pospiviroids in two samples. Of 10 cDNA clones analyzed, 9 were MPVd-like sequences and one was sequence of Tomato chlorotic dwarf viroid (TCDVd). Further analysis using full genomic sequences obtained by RT-PCR with previously designed primers (2) or a new set of primers (MTTVd-F: 5' GGG GAA ACC TGG AGC GAA CTG G, and MTTVd-R: 5' GGG GAT CCC TGA AGC GCT CCT) revealed genetic diversity in this population. Eight of thirteen cloned cDNAs represented by the 359-nt sequence of isolate Mex8 (GenBank Accession No. GQ131572) had 93 to 94% nucleotide sequence identity to other MPVd isolates (L78454 and L78456-L78463). Five other cDNA clones represented by the 361-nt sequence of isolate HM2 (GenBank Accession No. GQ131573) were 99% identical to a TCDVd isolate recently identified in Arizona (GenBank Accession No. FJ822878) and 96 to 97% identical to TCDVd isolates from other areas (GenBank Accession Nos. AF162131 and AB329668). These results are the first evidence of a mixed infection of two viroids infecting tomatoes in Mexico. MPVd was first identified in Mexico on papita (S. cardiophyllum) in 1996 (1). The origin of TCDVd in this greenhouse was not determined, but TCDVd potentially can be seed transmitted in tomato (3). The close relationship between the Mexican and the U.S. isolates suggests that TCDVd in these two countries may share a common origin, likely from seed. To our knowledge, this is the first report of a natural infection of MPVd and TCDVd on tomatoes in Mexico.

Identification of new variants of SCMV causing sugarcane mosaic in India and assessing their genetic diversity in relation to SCMV type strains

Sugarcane mosaic virus (SCMV) is one of the two causative viruses of mosaic in sugarcane, a sugar crop widely grown under tropical and subtropical conditions worldwide. Although molecular characterization of SCMV strains was reported from many countries, strains occurring in India, a major sugarcane producer have not been reported so far. Twenty-six sugarcane samples represented by seven major sugarcane growing states in India and USA were subjected to reverse-transcription polymerase chain reaction (RT-PCR) using a pair of newly designed coat protein specific primers. Among them 17 were found positive to the SCMV infection. The lengths of the sequences derived in this study using the new set of primers varied between 812 and 866 nt. The amino acid sequence comparison of 30 Indian SCMV isolates showed wide range of sequence similarities in core region (88.80-100%) and hyper variable region (51.3-100%). In the N-terminal region of the five Indian isolates, a deletion of 12 aa residues between aa 11 and 30 was observed, whereas the deletion was between aa 45 and 50 in SCMV-B and -D and between aa 61 and 70 in SCMV-A. The phylogenetic analyses performed with 46 SCMV CP sequences for both hyper variable region and core region separated the isolates mostly according to their geographical origin. The 30 Indian SCMV isolates were included exclusively in four groups besides SCMV-IND, which was grouped with SCMV-SC, a type of strain from Australia. Nearly 97.0% of the Indian isolates have no signs for close relationships with previously characterized SCMV type strains (SCMV-A, -B, -D, -E, and -SC) reported from other countries. Our studies revealed that the sugarcane mosaic in India are caused by at least nine new SCMV variants (IND-CC1, -CC2, -CC3, -CC4, -CO1, -CO2, -CP, -CS, and -J) and a type strain SCMV-SC represented by SCMV-IND. This is the first report on the variability and occurrence of new SCMV population in India.

No association of the TLR2 gene Arg753Gln polymorphism with rheumatic heart disease and Behçet’s disease

Behçet's disease (BD) is a multisystem inflammatory disorder of unknown etiology, and infections with different microorganisms including streptococci have been claimed as triggers of inflammatory attacks in BD pathogenesis. Toll-like receptor 2 (TLR2) has been known to recognize several microbial antigens including that of streptococci, and TLR2 gene Arg753Gln polymorphism has been reported to be strongly associated with acute rheumatic fever with an odds ratio of 100. This study aimed to investigate the TLR2 gene Arg753Gln polymorphism in a group of patients with BD and rheumatic heart disease (RHD) and to analyze the role of genotyping errors resulting from duplicated gene segments. The study group consisted of 211 patients with BD, 95 patients with RHD, and 94 matched Turkish healthy controls. Because of the duplicated exon 3 in 23-kb upstream of the TLR2 gene, genotyping for the Arg753Gln polymorphism with polymerase chain reaction-restriction fragment length polymorphism method was carried out using a new set of primers and PstI restriction enzyme. TLR2 gene Gln753 allele was observed in two of 211 (1.0%) patients with BD, five of 95 (5.3%) patients with RHD, and two of 94 (2.1%) healthy controls. All patients and controls were found to be heterozygous for Arg753Gln polymorphism, except one patient with BD, who was homozygous for Gln753. Although a slight increase of heterozygosity was noted in patients with RHD, no statistically significant difference was observed in the distribution of Arg753Gln polymorphism in BD and RHD compared to healthy controls. In conclusion, TLR2 gene Arg753Gln polymorphism is not associated with BD nor with RHD; and a duplicated region of the TLR2 exon 3 located 23-kb upstream of the polymorphic region may explain contradictory association findings described so far.

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn.