Ageing and environmental factors affect PCR success in wolf (Canis lupus) excremental DNA samples

Individual genotypes determined from noninvasive DNA samples (typically extracted from shed hairs or scats) are used to estimate population size in monitoring projects of elusive species. However, polymerase chain reaction (PCR) success rates usually are lower, and genotyping errors higher than in standard population genetic surveys, due to DNA degradation or contamination in aged field samples. In this study, we evaluate the results of common garden experiments showing that DNA degradation is significant in wolf (Canis lupus) scats older than 3 days, and it is enhanced in scats in direct contact with soil. A storage test showed that samples kept frozen in 95% ethanol performed better compared to other methods. However, variance of PCR success among samples was high, independent on sample age or storage condition. The detrimental consequences of DNA degradation can be avoided by collecting scat samples as fresh as possible, and implementing efficient multitube procedures and stringent quality control of the laboratory results. Efficient multitube procedures can produce reliable data, like in this study, which showed that the consensus genotypes obtained from excremental DNA exactly matched distinct reference genotypes obtained from wolf blood samples.     

An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (<Emphasis Type="Italic">Canis rufus</Emphasis>) experimental population area

Previously, sequencing of mitochondrial DNA (mtDNA) from non-invasively collected faecal material (scat) has been used to help manage hybridization in the wild red wolf (Canis rufus) population. This method is limited by the maternal inheritance of mtDNA and the inability to obtain individual identification. Here, we optimize the use of nuclear DNA microsatellite markers on red wolf scat DNA to distinguish between individuals and detect hybrids. We develop a data filtering method in which scat genotypes are compared to known blood genotypes to reduce the number of PCR amplifications needed. We apply our data filtering method and the more conservative maximum likelihood ratio method (MLR) of Miller et al. (2002 Genetics 160:357–366) to a scat dataset previously screened for hybrids by sequencing of mtDNA. Using seven microsatellite loci, we obtained genotypes for 105 scats, which were matched to 17 individuals. The PCR amplification success rate was 50% and genotyping error rates ranged from 6.6% to 52.1% per locus. Our data filtering method produced comparable results to the MLR method, and decreased the time and cost of analysis by 25%. Analysis of this dataset using our data filtering method verified that no hybrid individuals were present in the Alligator River National Wildlife Refuge, North Carolina in 2000. Our results demonstrate that nuclear DNA microsatellite analysis of red wolf scats provides an efficient and accurate approach to screen for new individuals and hybrids.     

Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes,L. 1758) populations

We investigated the feasibility of using genetic techniques to census pine marten (Martes martes) populations by genotyping non-invasively collected samples (plucked hair and scats), with particular reference to the genetically depauperate Irish population. Novel real-time polymerase chain reaction methods were developed for species and sex identification, targeting short DNA sequences. Background genetic variation at 17 microsatellite loci was very low in the Irish population, with an average of 2.29 alleles per locus and expected heterozygosity of 0.35. Despite such low polymorphism, a panel of eight loci with a sibling probability of identity of 0.011 reliably identified individual pine marten and their gender, as determined by reference to genotypes of live trapped individuals. With high nuclear DNA amplification success rates (93.8%) and low genotyping error rates (1.8%), plucked hairs may represent a more reliable and cost-effective DNA source than scats for monitoring populations of this elusive carnivore, and similar taxa such as the sympatric stone marten Martes foina.

     

Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post-deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey-based scats were more reliable when stored in EtOH than DET buffer. Insect-predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at ≥5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant-derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. © 2011 The Wildlife Society.     

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

Conservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.     

Population size estimation in Yellowstone wolves with error-prone noninvasive microsatellite genotypes

Determining population sizes can be difficult, but is essential for conservation. By counting distinct microsatellite genotypes, DNA from noninvasive samples (hair, faeces) allows estimation of population size. Problems arise because genotypes from noninvasive samples are error-prone, but genotyping errors can be reduced by multiple polymerase chain reaction (PCR). For faecal genotypes from wolves in Yellowstone National Park, error rates varied substantially among samples, often above the 'worst-case threshold' suggested by simulation. Consequently, a substantial proportion of multilocus genotypes held one or more errors, despite multiple PCR. These genotyping errors created several genotypes per individual and caused overestimation (up to 5.5-fold) of population size. We propose a 'matching approach' to eliminate this overestimation bias.     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.     

Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses

Noninvasive faecal DNA sampling has the potential to provide a wealth of information necessary for monitoring and managing endangered species while eliminating the need to capture, handle or observe rare individuals. However, scoring problems, and subsequent genotyping errors, associated with this monitoring method remain a great concern as they can lead to misidentification of individuals and biased estimates. We examined a kit fox scat data set (353 scats; 80 genotypes) for genotyping errors using both genetic and GIS analyses, and evaluated the feasibility of combining both approaches to assess reliability of the faecal DNA results. We further checked the appropriateness of using faecal genotypes to study kit fox populations by describing information about foxes that we could deduce from the 'acceptable' scat genotypes, and comparing it to information gathered with traditional field techniques. Overall, genetic tests indicated that our data set had a low rate of genotyping error. Furthermore, examination of distributions of scat locations confirmed our data set was relatively error free. We found that analysing information on sex primer consistency and scat locations provided a useful assessment of scat genotype error, and greatly limited the amount of additional laboratory work that was needed to identify potentially 'false' scores. 'Acceptable' scat genotypes revealed information on sex ratio, relatedness, fox movement patterns, latrine use, and size of home range. Results from genetic and field data were consistent, supporting the conclusion that our data set had a very low rate of genotyping error and that this noninvasive method is a reliable approach for monitoring kit foxes.     

A SNP chip to detect introgression in wildcats allows accurate genotyping of single hairs

Genotyping non-invasively collected samples is challenging. Nevertheless, genetic monitoring of elusive species like the European wildcat (Felis silvestris silvestris) mainly relies on such samples. Wildcats are likely threatened through introgression with domestic cats (F. silvestris catus). To determine introgression based on single cat hairs, we developed a 96.96 Fluidigm single nucleotide polymorphism (SNP) genotyping array chip. To estimate the accuracy of this method, we compared genotypes of 17 cats called with both Sanger sequencing and Fluidigm. When Sanger sequencing genotypes were considered as a reference, the genotyping error rate with Fluidigm was 0.9 %. We subsequently compared 16 hair samples to tissue samples of the same individual. When the tissue samples were used as a reference, the genotyping error rate in hair samples was 1.6 %. This low error rate allowed reliable recognition of individuals and correct assessment of introgression levels. Thus, the genotyping method presented in this paper is suitable for non-invasively collected samples. It will help conservationists to monitor the introgression rate in wildcat populations based on non-invasive hair sampling and subsequently to conduct effective conservation measures.     

Occurrence of selected genes of the Klebsiella pneumoniae clusters waa and wb for lipolysaccharide synthesis in reference and epidemic strains

The goal of presented study was to determine by PCR differences in existence or homology level of selected genes involved in K. pneumoniae lipopolysaccharide (LPS) synthesis and application of obtained results for genotyping. Number of 26 reference strains of K. pneumoniae belonging to serogroups O1, O2a, O2a2e, O2a2e2h, O2a2f2g, O3, O4, O5, O7, O8, and O12 was tested together with 13 epidemic strains from 5 outbreaks and 6 casual isolates for the existence of 7 (waaA, waaE, waaL, waaQ, waaZ, waaX and uge) and 4 (wbdA, wbdC, manB, wbbO) genes of the waa and wb clusters for LPS biosynthesis. Based on PCR results, 10 and 11 genotypes were distinguished in tested strains for genes from waa and wb clusters respectively. Derived dendrograms were topologically dissimilar, however observed correlation between clonal groups and O-group was marginal for both compared clusters. Since we aimed to develop genotyping method for K. pneumoniae, genes from clusters waa and wb were used together to enhance the distinguishing capacity. Twenty-one genotypes were distinguished in 45 tested strains (DI=0,46) when 11 genes were applied for typing. Although no apparent correlation between genotype and serogroup was observed, epidemic isolates from 5 outbreaks were diversified into 5 genotypes, whereas strains from the same outbreak were indistinguishable. Described here genotyping method is determinative and was found time and cost effective. This method may be applied in every clinical laboratory equipped in an ordinary PCR apparatus.     

Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies

We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy.     

High-resolution melting molecular signatures for rapid identification of human papillomavirus genotypes

Background

Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes.

Methods

Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates.

Results

A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215–221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.

Conclusions

This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner.     

Determination of the optimal tissue source and number of microsatellites for detection of zygotic origin of cattle twins

Abstract

Twenty pairs of cattle twins were genotyped for 3 to 12 microsatellites each using semen, blood, milk and hair roots. Chimaerism was recognized in 19 pairs by discrepancies in microsatellite analysis from milk and blood as opposed to semen and hair, or by detection of more than 2 alleles per genotype from milk and blood. Chimaerism of 2, 3 or 4 alleles was demonstrated in genotypes of twins from blood as compared to 1 or 2 alleles only from hair. The appearance of predominant bands in genotypes from blood or milk representing alleles of only one of the co‐twins was not consistent among the different microsatellites for the same twins. No evidence for germ cell chimaerism was found in semen of dizygotic male twins although our PCR system could detect cell mixes as small as a 1:100 ratio. Genotyping from either hair or semen for 4 microsatellites are sufficient to confirm zygotic origin of twins at .98 accuracy. Researchers should be aware of the possibility of erroneous genotyping when analyzing DNA from twins derived from blood or milk and the potential of chimaerism as an experimental model to study different immunological characteristics of cattle co‐twins.     

TECHNICAL ADVANCES: Effects of genotyping protocols on success and errors in identifying individual river otters (Lontra canadensis) from their faeces

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method.     

PCR fingerprinting for epidemiological studies of Staphylococcus aureus

Staphylococcus aureus isolates (n = 126), collected during two different periods from patients hospitalised in pediatric wards, were analysed using polymerase chain reaction (PCR) mediated genotyping. These isolates were compared with 29 isolates from individuals attending the out-patient clinic of the same hospital and 13 isolates from pediatric hospital personnel. Within a group of 99 isolates gathered from 48 individuals during surveillance period I, 22 distinct genotypes were identified by application of two PCR assays. Among the 58 isolates collected in surveillance period II from pediatric and out-clinic patients, 25 genotypes were detected by a single PCR assay only. Based on these results it was demonstrated that patients can be colonised with multiple strains that may persist in a certain anatomical location for prolonged periods of time. It is shown that persistence of a S. aureus strain in a pediatric ward can be deduced from the PCR genotyping studies. As such PCR can be used for longitudinal monitoring of bacterial infections in hospital departments, analysis of patient-to-patient and personnel-to-patient transmission and for detection of genetic variation in general in S. aureus. Also, isolate-specific DNA probes can be generated for S. aureus by PCR genotyping. The probes can be used for the recognition of re-emerging S. aureus epidemics.     

A method of oligochip for single nucleotide polymorphism genotyping in the promoter region of the interleukin-1 beta gene and its clinical application

Single nucleotide polymorphisms (SNPs) can contribute to genetic predispositions or serve as genetic markers that are associated with complex diseases. So far, a few SNP arrays containing a limited number of SNPs have been used in routine genetic testing. This study described an oligochip-based method that genotypes two SNPs (-511 and -31) in the promoter region of the interleukin (IL)-1 beta gene. The sensitivity of this SNP genotyping method is derived from polymerase chain reaction (PCR)-amplified allele-specific primer-probes with a biotin label incorporated from the reverse primers. The amplified primer-probes can specifically hybridize with the oligonucleotides that are spotted on the oligochip. This oligochip-based method successfully discriminated the two biallelic SNPs with 9 different genotypes and all the genotyping results are in concordance with those from PCR restriction fragment length polymorphism (RFLP) analysis. Selective samples with various genotypes were also confirmed by direct sequencing. This method was applied in the genotyping of the patients with tuberculosis or gastric cancer and healthy controls. In the case control study, our genotyping data supported the reported association between gastric cancer and the genotypes of IL-1 beta -31 TT and -511 CC (p < 0.05). We also found that there is a significant difference of IL-1 beta -31 genotypes between 98 tuberculosis patients and healthy controls (p < 0.002). All of our results demonstrated that the oligochip can effectively and accurately identify SNP genotypes in the IL-1 beta promoter region.     

Multiplex pre-amplification for noninvasive genetic sampling: is the extra effort worth it?

Microsatellite genotyping of hair and faeces using standard polymerase chain reaction (PCR) resulted in low success rates and high error rates in a 2003–2004 pilot study using noninvasive genetic sampling for the brown bear (Ursus arctos) in the Italian Alps. Thus, we evaluated the performance of multiplex pre-amplification for improving microsatellite genotyping results. Brown bear faecal DNA extracts of varying quality (n = 33) and hair DNA extracts of poor (n = 32) and good (n = 34) quality were used to compare standard PCR and pre-amplification. In contrast to previous studies, there was no significant difference between methods for individual locus amplification success, genotyping error and genotyping success rates for scat and hair samples. The use of pre-amplification requires an additional investment of time and resources, and our results raise questions about the universal value of pre-amplification approaches. We suggest that researchers carefully evaluate the performance of pre-amplification compared to standard PCR using field-collected samples from the study area of interest before engaging in large-scale noninvasive genetic analyses.     

Biological and environmental degradation of gorilla hair and microsatellite amplification success

Naturally shed hairs are an important source of genetic material for both conservation and forensics but are notoriously poor sources of DNA. DNA degradation in hair roots is caused by apoptosis as part of the cycle of hair growth and by autolysis in decomposing animals. Shed hairs are additionally exposed to degenerative environmental processes. However, genetic studies rarely examine hair root morphologies or refer to root growth phases prior to analysis, and detailed knowledge of the rapidity of DNA degradation amongst shed hairs is lacking. We examined the effects of biological and environmental processes on western lowland gorilla ( Gorilla gorilla gorilla Savage and Wyman) hair roots with respect to morphological characteristics and polymerase chain reaction (PCR) success at eight nuclear loci. Root type frequencies indicate that gorilla body hairs may exhibit a longer telogen phase than human head hairs. All plucked hair root types amplified more efficiently than shed hairs, and only 41% of shed hairs had root types considered suitable for genotyping. Telogen hairs from fresh nests were four-fold more useful for genotyping if the roots were associated with translucent epithelial tissue, and preselection of these root types doubled the overall data-yield to 58%. Nest age correlated with root morphology and PCR success, and PCR success was almost halved after 3 days of exposure. Finally, an association between postmortem interval, root morphology, and PCR success was observed that was consistent with postmortem changes reported in human head hairs.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 281–294.     

采用PCR-测序法对北京地区325例妇女宫颈脱落细胞样品中人乳头瘤病毒进行检测和基因分型

为了探讨PCR-测序法在宫颈脱落细胞样品中人乳头瘤病毒 (Human papillomavirus, HPV) 临床检测中的应用价值,采用HPV通用引物PGMY09/11针对HPV L1区基因序列进行PCR扩增,并通过DNA测序法对HPV进行基因分型。对于混合感染样品,利用HPV型别特异性引物PCR的方法进行基因分型。325例临床样品中,228例为HPV阳性,其中66例为混合感染。共发现27种不同的HPV型别,其中HPV 16比例最多,其次是HPV 58和52。高危型HPV检出率随病变程度加重显著性增加     

Impacts of sampling location within a faeces on DNA quality in two carnivore species

We investigated the influence of sampling location within a faeces on DNA quality by sampling from both the outside and inside of 25 brown bear (Ursus arctos) scats and the side and the tip of 30 grey wolf (Canis lupus) scats. The outside of the bear scat and side of the wolf scat had significantly lower nuclear DNA microsatellite allelic dropout error rates (U. arctos: P = 0.017; C. lupus: P = 0.025) and significantly higher finalized genotyping success rates (U. arctos: P = 0.017; C. lupus: P = 0.012) than the tip and inside of the scat. A review of the faecal DNA literature indicated that <45% of studies report the sampling location within a faeces indicating that this methodological consideration is currently underappreciated. Based on our results, we recommend sampling from the side of canid scats and the outside portion of ursid scats to obtain higher quality DNA samples. The sampling location within a faeces should be carefully considered and reported as it can directly influence laboratory costs and efficiency, as well as the ability to obtain reliable genotypes.