Multiplex PCR Sets Research Articles

Molecular characterization of carbapenem-resistant Acinetobacter baumannii clinical isolates from Egyptian patients.

Acinetobacter baumannii (A. baumannii) represents a global threat owing to its ability to resist most of the currently available antimicrobial agents. Moreover, emergence of carbapenem resistant A. baumannii (CR-AB) isolates limits the available treatment options. Enzymatic degradation by variety of ß-lactamases, have been identified as the most common mechanism of carbapenem resistance in A. baumannii. The alarming increase in the prevalence of CR-AB necessitates continuous screening and molecular characterization to appreciate the problem. The present study was performed to assess the prevalence and characterize carbapenemases among 206 CR-AB isolated from various clinical specimens collected from different intensive care units at Kasr Al-Aini Hospital. All isolates were confirmed to be A. baumannii by detection of the blaOXA-51-like gene. Molecular screening of 13 common Ambler class bla carbapenemases genes in addition to insertion sequence (IS-1) upstream OXA-23 were performed by using four sets of multiplex PCR, followed by identification using gene sequencing technology. Among the investigated genes, the prevalence of blaOXA-23, and blaOXA-58 were 77.7%, and 1.9%, respectively. The ISAba1 was detected in 10% of the blaOXA-23 positive isolates. The prevalence of metallo-β-lactamases (MBLs) studied; blaNDM-1, blaSPM, blaVIM, blaSIM-1 were 11.7%, 6.3%, 0.5%, and 0.5% respectively. One of class A; bla KPC was detected in 10.7% of the investigated isolates. blaOXA-24/40, blaIMP, blaGES, blaVEB and blaGIM were not detected in any of the studied isolates. Moreover, 18.4% of the isolates have shown to harbor two or more of the screened bla genes. We concluded that the most prevalent type of ß-lactamases genes among CR-AB isolates collected from Egyptian patients were blaOXA-23 followed by blaNDM-1 and blaKPC.

Carbapenem Resistance Profiles of Pathogenic Escherichia coli in Uganda

Escherichia coli has been implicated as one of the main etiological agents of diarrhea, urinary tract infections, meningitis and septicemia worldwide. The ability to cause diseases is potentiated by presence of virulence factors. The virulence factors influence the capacity of E. coli to infect and colonize different body systems. Thus, pathogenic E. coli are grouped into DEC strains that are mainly clustered in phylogenetic group B1 and A; ExPEC belonging to A, B2 and D. Coexistence of virulence and beta-lactamase encoding genes complicates treatment outcomes. Therefore, this study aimed at presenting the carbapenem resistance (CR) profiles among pathogenic E. coli. This was a retrospective cross-sectional study involving use of 421 archived E. coli clinical isolates collected in 2019 from four Uganda tertiary hospitals. The isolates were subjected to antibiotics sensitivity assays to determine phenotypic resistance. Four sets of multiplex PCR were performed to detect CR genes, DEC pathotypes virulence genes, ExPEC PAI and the E. coli phylogenetic groups. Antibiotic susceptibility revealed that all the 421 E. coli isolates used were MDR as they exhibited 100% resistance to more than one of the first-line antibiotics. The study registered phenotypic and genotypic CR prevalence of 22.8% and 33.0% respectively. The most predominant gene was blaOXA-48 with genotypic frequency of 33.0%, then blaVIM (21.0%), blaIMP (16.5%), blaKPC (14.8%) and blaNDM (14.8%). Spearman’s correlation revealed that presence of CR genes was highly associated with phenotypic resistance. Furthermore, of 421 MDR E. coli isolates, 19.7% harboured DEC virulence genes, where EPEC recorded significantly higher prevalence (10.8%) followed by S-ETEC (3.1%), STEC (2.9%), EIEC (2.0%) and L-ETEC (2.0%). Genetic analysis characterized 46.1% of the isolates as ExPEC and only PAI IV536 (33.0%) and PAI IICFT073 (13.1%) were detected. Phylogenetic group B2 was predominantly detected (41.1%), followed by A (30.2%), B1(21.6%), and D (7.1%). Furthermore, 38.6% and 23.1% of the DEC and ExPEC respectively expressed phenotypic resistance. Our results exhibited significant level of CR carriage among the MDR DEC and ExPEC clinical isolates belonging to phylogenetic groups B1 and B2 respectively. Virulence and CR genetic factors are mainly located on mobile elements. Thus, constitutes a great threat to the healthcare system as this promotes horizontal gene transfer.

Multiplex PCR Assays for the Detection of One Hundred and Thirty Seven Serogroups of Shiga Toxin-Producing Escherichia coli Associated With Cattle.

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called “non-top-7” have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7–12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

Multiplex PCR Sets of Novel Microsatellite Loci for Iwagaki Oyster Crassostrea nippona and Their Application in Parentage Assignment

Iwagaki oyster, Crassostrea nippona, widely distributes along the seashore of Eastern Asia, and has been considered to be a potential breeding species due to its delicious taste and high commercial value. In order to study its genetic background and population structure, we developed 46 novel polymorphic microsatellite markers using next-generation sequencing technique and characterized them in 30 individuals. The number of alleles ranged from 3 to 22, while the observed and expected heterozygosities varied from 0.133 to 1.000 and 0.455 to 0.949, respectively. Fifteen microsatellite markers were selected and grouped into five highly informative multiplex PCRs for C. nippona. We evaluated and validated these multiplex PCRs in a cultured population including 173 candidate parents and 486 offspring. In actual parentage analysis, 80% of the offspring were correctly assigned to their parental pairs using three multiplex PCRs. Furthermore, the success rate of parentage assignment reached 96% when the other two multiplex PCRs were added. These 46 microsatellite loci with high variability and the five multiplex PCRs described here provide a powerful tool for pedigree reconstruction, resource conservation and selective breeding program of C. nippona.

Mutational spectrum of autosomal recessive limb-girdle muscular dystrophies in a cohort of 112 Iranian patients and reporting of a possible founder effect

BackgroundLimb-girdle muscular dystrophies are a group of genetically heterogeneous diseases that are inherited in both autosomal dominant (LGMDD) and autosomal recessive forms (LGMDR), the latter is more common especially in populations with high consanguineous marriages like Iran. In the present study, we aimed to investigate the genetic basis of patients who are suspicious of being affected by LGMDR.DNA samples of 60 families suspected of LGMD were extracted from their whole blood. Four short tandem repeat (STR) markers for each candidate genes related to LGMD R1 (calpain3 related)- R6 (δ-sarcoglycan-related) were selected, and all these 24 STRs were applied in two sets of multiplex PCR. After autozygosity mapping, Sanger sequencing and variant analysis were done. Predicting identified variants’ effect was performed using in-silico tools, and they were interpreted according to the American College of Medical Genomics and Genetics (ACMG) guideline. MLPA was used for those patients who had large deletions.Fresh muscle specimens were taken from subjects and were evaluated using the conventional panel of histochemical stains.Resultsforty out of sixty families showed homozygote haplotypes in CAPN3, DYSF, SGCA, and SGCB genes. The exons and intron-exon boundaries of the relevant genes were sequenced and totally 38 mutations including CAPN3 (n = 15), DYSF (n = 9), SGCB (n = 11), and SGCA (n = 3) were identified. Five out of them were novel. The most prevalent form of LGMDs in our study was calpainopathy followed by sarcoglycanopathy in which beta-sarcoglycanopathy was the most common form amongst them. Exon 2 deletion in the SGCB gene was the most frequent mutation in this study.We also reported evidence of a possible founder effect in families with mutations in DYSF and SGCB genes. We also detected a large consanguineous family suffered from calpainopathy who showed allelic heterogeneity.ConclusionsThis study can expand our knowledge about the genetic spectrum of LGMD in Iran, and also suggest the probable founder effects in some Iranian subpopulations which confirming it with more sample size can facilitate our genetic diagnosis and genetic counseling.

Molecular typing of Streptococcus agalactiae isolates of serotype Ia from tilapia in southern China.

Streptococcus agalactiae is an important pathogen of tilapia causing enormous economic losses worldwide. In this study, multilocus sequence typing indicated that 75 S. agalactiae isolates from tilapia in southern China belonged to sequence type-7, as well as belonging to serotype Ia, as confirmed by multiplex PCR assay. The putative-virulence gene profiles and genetic variation of these strains were determined by three sets of multiplex PCR and multi-virulence locus sequencing typing (MVLST), respectively. Analysis of putative-virulence gene profiles showed that each strain harbored 18 putative-virulence genes but lacked lmb and scpB. Three putative-virulence genes (srr-1, bibA and fbsA) were further selected for MVLST analysis. Our data showed that the strains had 14 MVLST types (1-14) and clustered in three groups (Groups I-Ⅲ). The period of time during 2013 and 2014 was an important turning point for the differentiation of the putative-virulence genes of S. agalactiae, as type 1 within Group Ⅱ became the predominant MVLST type. There were significant differences in MVLST types of S. agalactiae isolated from different tilapia farming regions. MVLST assay may improve the discriminatory power and is suitable for understanding the epidemiology of S. agalactiae serotype Ia and screening multivalent vaccine candidate strains.

Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0–14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0–14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

Detection of Vibrio and ten Vibrio species in cage-cultured fish by multiplex polymerase chain reaction using house-keeping genes

Vibriosis is one of the major bacterial diseases observed in cultured marine fish worldwide. An epidemic trait of the Vibrio derived from cage-cultured marine fish is still lacking. To identify the Vibrio and discriminate ten Vibrio species from cage-cultured marine fish, we designed specific primers targeting the RNA polymerase sigma factor (rpoD), recombination protein F (recF), transcriptional regulator (toxR) genes (known as housekeeping genes), and two multiplex PCR sets (PCR set 1 for V. anguillarum, V. gigantis, V. atlanticus, V. harveyi, and V. scophthalmi; and PCR set 2 for V. lentus, V. splendidus, Photoacterium piscicola, V. alginolyticus and P. damselae). Moreover, each multiplex PCR assay comprised internal control primers that enabled the detection of the Vibrio. Designed primers yielded specific diagnostic results to target the Vibrio and ten Vibrio species in the multiplex PCR assays. The minimal detection limits of the multiplex PCR were each 5 ng DNA μL−1, which showed ten-fold sensitivity compared to a single PCR reaction. The novel multiplex PCR assays could contribute to detecting the Vibrio and discriminate the major Vibrio species in cage-cultured marine fish, and could be used as a reliable and informative diagnostic tool in the aquaculture industry.

Parentage Assignment in Striped Catfish (Pangasianodon hypophthalmus) Based on Microsatellite Marke

In this study, five microsatellite markers from two multiplex PCR sets were used to determine the pedigree of five striped catfish (Pangasianodon hypophthalmus) families that came from a breeding program in Vietnam. The number of alleles per locus and polymorphic information content (PIC) were 4 to 7 and 0.551-0.803, respectively. In the parentage analysis based on five microsatellites, 62.7% of offspring could be allocated unambiguously to their parental pairs using the perfect mismatch setting, and assignment rate improved to 89.3% after a single mismatch was allowed. Parentage analysis when only four microsatellites were used (a locus with a high frequency of null alleles was excluded) increased the assignment rate to 68% for a perfect mismatch and to 90% for assignment with a single mismatch. Results here confirm that a DNA markerbased approach to parentage assignment in P. hypophthalmus will be useful in genetic improvement programs for this important aquaculture species in Vietnam. .

Validation of 12 species-specific, tetrasomic microsatellite loci from the Russian sturgeon, Acipenser gueldenstaedtii, for genetic broodstock management

The Russian sturgeon, Acipenser gueldenstaedtii, is a critically endangered fish species. Hatcheries are operated in several countries within its natural range to produce stocking material for release into the wild and also for aquaculture purposes (caviar and meat production). An appropriate genetic broodstock management (plan or strategy) is required to avoid negative effects, e.g., admixture and hybridization of genetically differing stocks or loss of genetic variability due to inbreeding and genetic drift. Therefore, 11 tetrasomic microsatellite loci were newly isolated from the Russian sturgeon genome and arranged together with an already known locus into four multiplex PCR sets. These microsatellites were used to characterize three groups of hatchery juveniles from Germany (aquaculture production), Turkey, and Romania (production of stocking material) as well as a group of wild-caught adults from the Danube River, Romania. Based on the variability within groups, measured by the mean number of alleles per locus and expected heterozygosity, and the differentiation between groups, measured by Nei’s GST and genetic distance D, the ability of the 12 loci to detect unwanted reductions in genetic variability within hatchery juveniles and to differentiate between groups could be demonstrated. This set of loci can also be used to identify those pairs of spawners that transmit the highest possible genetic variability to the next generation.

Isolation and characterization of 18 polymorphic microsatellite markers for the critically endangered stellate sturgeon, Acipenser stellatus

The stellate sturgeon, Acipenser stellatus, is a critically endangered fish species. Knowledge on its genetic diversity and population structure is urgently needed to enable the identification of management units in order to prevent extinction. Therefore, 18 species-specific, polymorphic microsatellite loci have been isolated using GS-FLX Titanium pyrosequencing, arranged into 6 multiplex PCR sets, and characterized in 52 individuals (20 farmed and 32 wild). The total number of alleles per locus varied between 3 and 36 with an average of 8.44. The wild individuals were more diverse with an average number of 8.17 alleles per locus than the farmed ones with 3.28 alleles per locus. Observed heterozygosities ranged from 0.050 to 0.950 in the farmed and from 0.094 to 0.969 in the wild individuals. Significant deviations from Hardy-Weinberg equilibrium were found at 3 loci of the farmed and 5 loci of the wild individuals. The two sturgeon groups were significantly differentiated (F ST = 0.118). The high sensitivity and discriminatory power of the 18 loci were proven by a very low overall probability of identity for siblings (PIsib = 8.73 × 10−6) and a high accuracy of self-classification (98%). Thus, these newly developed markers represent a valuable genetic toolbox to identify management units for species conservation and sustainable fisheries.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii in a Tertiary Care Hospital in Egypt: Clonal Spread of blaOXA-23

Of great concern is the increased frequency of carbapenem-resistant Acinetobacter baumannii (CRAB) causing healthcare-associated infections. Different classes of β-lactamases are involved in this resistance through hydrolyzing carbapenems. Multilocus sequence typing (MLST) has been applied successfully for characterizing different varieties of bacterial pathogens epidemiologically. In the present study, we aimed to type and characterize the resistance profile of clinical isolates of CRAB causing healthcare-associated infections in patients admitted to Kasr Al-Aini hospital, using MLST, and compare with sequence types (STs) from other countries. A total of 50 isolates were collected from clinical samples (predominantly wound and blood), then identified by blaOXA-51-like gene PCR, and subjected to Oxford MLST scheme. The ST was designated according to PubMLST database, and e-BURST algorithm was used to assign clonal complexes. Four sets of multiplex PCR were performed to detect common carbapenem resistance genes. ST391 was the predominant ST detected in 17 cases, 70.5% of which harbored blaOXA-23 alone, both blaOXA-23 and blaKPC in 11.8%. Newly recognized 13 STs were submitted to the PubMLST database. Carbapenem resistance due to blaOXA-23 carbapenemase was detected in 36/50 (72%), followed by blaOXA-23 concomitant with blaKPC in 7/50 (14%), while blaNDM with blaOXA-58 in 3/50 (6%) and blaNDM alone in 1 case (2%). To conclude, this study demonstrates the propagation of highly resistant clone of STs 391 and 1151, carrying blaOXA-23 genes, with the first report of blaKPC in blaOXA carrying CRAB and the presence of new STs by performing the MLST technique in an Egyptian laboratory facility.

Four multiplex PCR Sets of 11 LM Maize for LMO environmental monitoring in Korea

Four multiplex PCR Sets of 11 LM Maize for LMO environmental monitoring in Korea

A multiplex PCR system for 13 RM Y-STRs with separate amplification of two different repeat motif structures in DYF403S1a

In forensic science and human genetics, Y-chromosomal short tandem repeats (Y-STRs) have been used as very useful markers. Recently, more Y-STR markers have been analyzed to enhance the resolution power in haplotype analysis, and 13 rapidly mutating (RM) Y-STRs have been suggested as revolutionary tools that can widen Y-chromosomal application from paternal lineage differentiation to male individualization. We have constructed two multiplex PCR sets for the amplification of 13 RM Y-STRs, which yield small-sized amplicons (<400bp) and a more balanced PCR efficiency with minimum PCR cycling. In particular, with the developed multiplex PCR system, we could separate three copies of DYF403S1a into two copies of DYF403S1a and one of DYF403S1b1. This is because DYF403S1b1 possesses distinguishable sequences from DYF403S1a at both the front and rear flanking regions of the repeat motif; therefore, the locus could be separately amplified using sequence-specific primers. In addition, the other copy, defined as DYF403S1b by Ballantyne et al., was renamed DYF403S1b2 because of its similar flanking region sequence to DYF403S1b1. By redefining DYF403S1 with the developed multiplex system, all genotypes of four copies could be successfully typed and more diverse haplotypes were obtained. We analyzed haplotype distributions in 705 Korean males based on four different Y-STR subsets: Yfiler, PowerPlex Y23, Yfiler Plus, and RM Y-STRs. All haplotypes obtained from RM Y-STRs were the most diverse and showed strong discriminatory power in Korean population.

A new suite of twenty-two polymorphic microsatellite loci in the parasitic wasp, Habrobracon hebetor (Hymenoptera: Braconidae): Promising molecular tools for studying the population genetics of several beneficial braconid species

Combining a biotin-enrichment protocol and 454GS-FLX titanium pyrosequencing technology, we characterised 22 polymorphic microsatellite loci from the parasitic wasp, Habrobracon hebetor (Say) (Hymenoptera: Braconidae), a cosmopolitan species commonly used in biological control against a wide range of both major lepidopterous pests of stored products and field crops in different parts of the world. Three multiplex PCR sets were optimised and characterised across 46 H. hebetor specimens from two samples collected from millet fields in Niger. Two to 11 alleles were found per locus and observed heterozygosity ranged from 0.289 to 0.826. Polymorphism was detected in both samples with a similar level of observed heterozygosity (0.482 vs. 0.502) and number of alleles (4.1 vs. 3.6). Deviation from Hardy-Weinberg equilibrium was detected at the same five loci in both samples and five or seven more loci in each sample but was not associated with heterozygote deficiencies. Even though evidence for linkage disequilibrium was found between a few alleles, these new loci segregated independently. The variability of the 22 loci will enable estimates of genetic diversity and structure patterns, as well as gene flow between H. hebetor populations at different spatial scales. Cross-species amplifications were successful among the six Bracon spp. tested and nine loci will be particularly appropriate for population genetic studies in B. brevicornis.

Cross-species amplification of microsatellite loci for non-invasive genetic monitoring of the snowy owl (Bubo scandiacus)

Genetic analysis has shown to be an important and efficient method for monitoring species, but the lack of genetic markers constrains such monitoring for many species. Here, we cross-amplified microsatellite loci in the snowy owl (Bubo scandiacus) using primers previously characterized in other species. Among polymorphic loci, we selected 12 loci that amplified relatively short fragments (<250 base pairs) to facilitate genotyping of moulted feathers and other non-invasively collected samples. These 12 loci, and a sex-typing marker, were amplified in two multiplex PCR sets and used to screen 49 snowy owls from a northern European population. The number of alleles observed at single loci was 2 to 17. Expected and observed heterozygosity ranged from 0.12 to 0.91 and from 0.12 to 0.96, respectively. The combined probability of identity for the 12 loci was 5.7 × 10−11. Our multiplex PCR assays are expected to be useful for genetic monitoring, parentage analysis, and population genetic studies of the snowy owl.

Primer Evaluation for PCR and its Application for Detection of Carbapenemases in Enterobacteriaceae.

Background:During the last decade, the prevalence of carbapenem-resistant Enterobacteriaceae in human patients has increased. Carbapenemase-producing bacteria are usually multidrug resistant. Therefore, early recognition of carbapenemase producers is critical to prevent their spread.Objectives:The objective of this study was to develop the primers for single and/or multiplex PCR amplification assays for simultaneous identification of class A, class B, and class D carbapenem hydrolyzing β-lactamases in Enterobacteriaceae and then to evaluate their efficiency.Materials and Methods:The reference sequences of all genes encoding carbapenemases were downloaded from GenBank. Primers were designed to amplify the following 11 genes: blaKPC, blaOXA, blaVIM, blaNDM, blaIMP, blaSME, blaIMI, blaGES, blaGIM, blaDIM and blaCMY. PCR conditions were tested to amplify fragments of different sizes. Two multiplex PCR sets were created for the detection of clinically important carbapenemases. The third set of primers was included for detection of all known carbapenemases in Enterobacteriaceae. They were evaluated using six reference strains and nine clinical isolates.Results:Using optimized conditions, all carbapenemase-positive controls yielded predicted amplicon sizes and confirmed the specificity of the primers in single and multiplex PCR.Conclusions:We have reported here a reliable method, composed of single and multiplex PCR assays, for screening all clinically known carbapenemases. Primers tested in silico and in vitro may distinguish carbapenem-resistant Enterobacteriaceae and could assist in combating the spread of carbapenem resistance in Enterobacteriaceae.

Population database on: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D22S1045, D12S391, D16S539, D18S51, D19S433, D21S11, FGA, TH01, vWA loci included in NGM system based on one thousand unrelated individuals from Lodz region of Central Poland

A population data obtained on the basis of sample of 1000 unrelated individuals of Polish ancestry living in Lodz region of Central Poland with use of fluorescent multiplex-PCR and capillary electrophoresis were presented. Evaluation included 15 polymorphic loci DNA - STR from NGM multiplex-PCR set, ie. D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA. The allele frequency distribution and crucial statistical parameters for the investigated markers and the whole set were calculated. The compliance of the studied population with Hardy-Weinberg equilibrium, independence of inheritance and high parameters of the usefulness in forensic genetics have been demonstrated. The interpopulation comparison performed by the "neighbor-joining" method as well as multidimensional scaling depicted the genetic distances dividing the examined Polish population from other populations of Poland, Europe and the world.

Isolation, characterization and multiplex PCR development of Bean Goose (Anser fabalis) microsatellite loci

DNA from non-invasively collected samples may be used to identify individuals for monitoring birds. Here we describe 36 novel microsatellite loci from Bean Goose (Anser fabalis) identified using a next-generation sequencing approach. From 34 variable loci, we selected 12 tetranucleotide loci with short amplicon sizes, combined these into two multiplex PCR sets, and included a sex-typing marker. In 31 Bean Geese from a population in central Norway, we found 4–10 alleles per locus and observed heterozygosity ranging from 0.58 to 0.87. The combined probability of identity for the 12 loci was 6.5 × 10−13. These novel microsatellite loci and their multiplex PCR assays will be useful for genetic analyses of the Bean Goose, including typing of non-invasive samples such as molted feathers. Cross-species application of the two multiplex PCR assays revealed that all 12 loci amplified successfully in four other Anser species.

A multiplex microsatellite set for non-invasive genotyping and sexing of the osprey (Pandion haliaetus).

During the 1950s and 1970s the osprey (Pandion haliaetus) experienced a dramatic population crash and remains of conservation concern in several parts of the world. We isolated 37 microsatellite loci and assessed these in ospreys sampled in the UK and Norway (using mouth swabs/feathers). From 26 loci variable in four ospreys, we selected 13, combined these into two multiplex-PCR sets and included a sex-typing marker. Additional markers confirmed sexes. In 17 ospreys, feather-sampled in central Norway, we found 3–10 alleles per locus. The 13 loci are autosomal (heterozygotes were present in both sexes) and observed heterozygosities ranged from 0.24 to 0.94. The combined probability of identity for the 13 loci was 8.0 × 10−12. These microsatellite loci will be useful for genetic monitoring, parentage analysis and population genetic studies of the osprey.Electronic supplementary materialThe online version of this article (doi:10.1007/s12686-015-0497-4) contains supplementary material, which is available to authorized users.