Non-invasive PCR Research Articles

Polymerase Chain Reaction on In-cage Filter Paper at Different Time Points to Detect Helicobacter spp.

Helicobacter spp. infections in mice can have broad-ranging effects on gastrointestinal, reproductive, and immune systems. This can introduce significant confounding variables for research and may reduce scientific rigor. Screening mouse colonies for Helicobacter species can be accomplished via noninvasive PCR testing on filter paper placed in animal-free dirty bedding sentinel cages. In our facility, one tablespoon of dirty bedding from each cage on a rack is added to a designated sentinel cage every 3 wk at cage change, and PCR testing is performed on in-cage filter paper quarterly. We hypothesized that cages that received Helicobacter spp.-positive bedding at later time points would have a lower detection rate of Helicobacter spp. with PCR testing compared with cages that received positive bedding at earlier time points due to the filter paper becoming saturated. To determine if screening would be able to detect one positive row of cages on a rack, 9 tablespoons of Helicobacter- positive bedding and 71 tablespoons of negative bedding were added at the 3-, 6-, or 9-wk time points to 14 empty sentinel cages per time point. Negative bedding was added every 3 wk to cages not scheduled to receive positive bedding. Negative controls received 80 tablespoons of negative bedding and positive controls received 80 tablespoons of positive bedding at each time point. Filter paper was tested via PCR for Helicobacter spp. at 12 wk. All positive controls tested positive, and all negative controls tested negative. Two 3-wk cages, two 6-wk cages, and three 9-wk cages were positive, indicating no difference between time points. This resulted in a 16.7% Helicobacter spp. detection rate. These results indicate that PCR on in-cage filter paper may not be reliable in detecting low levels of Helicobacter spp. nucleic acid in dirty bedding.

New Strategy for the Detection and Treatment of Helicobacter pylori Infections in Primary Care Guided by a Non-Invasive PCR in Stool: Protocol of the French HepyPrim Study.

Helicobacter pylori (Hp) infects half of the world population and is responsible for gastric, duodenal ulcers and gastric cancer. The eradication of Hp cures ulcers and prevents ulcer recurrences and gastric cancer. Antibiotic resistance of Hp, and particularly clarithromycin resistance, is the primary cause of treatment failure and is a major concern identified by the WHO as a high priority requiring research into new strategies. Treatments guided by the detection of antibiotic resistance have proven their medical and economical superiority. However, this strategy is severely hampered by the invasive nature of the fibroscopy, since antibiotic resistance detection requires gastric biopsies. The eradication of Hp involves primary care physicians. The objective of this study will be to evaluate the feasibility of a strategy for the management of Hp infection in primary care by a recently developed non-invasive procedure and its non-inferiority in eradication rates compared with the strategy recommended by the French National Authority of Health. The non-invasive procedure is a PCR on stool to detect Hp infection and mutations conferring resistance to clarithromycin allowing a treatment guided by the results of the PCR. We present the protocol of a prospective, multicenter, randomized, controlled interventional study in two arms.

Sex identification based on the CHD gene from Gentoo penguin (Pygoscelis papua) fecal DNA samples

We developed a reliable noninvasive PCR method for sex identification of the Gentoo penguin (Pygoscelis papua) based on the amplification of the chromobox-helicase DNA-binding (CHD) gene. Two sets of nested primers (F1/R1, F2/R2) were designed to amplify the introns of the CHD1W and CHD1Z genes. The fecal samples were lysed in an optimized lysis reagent containing PCR buffer. The fecal lysate was employed as the template DNA to perform PCR amplification. Following nested PCR, agarose gel electrophoresis was performed, and the amplicons exhibited a sex-specific band pattern (211 bp and 315 bp in females, 211 bp in males). There was no amplification failure of our specific primers in 14 tested samples (7 males and 7 females), and the results of genotypic identification matched the records of sex based on oviposition behavior and pairing combination (based on the monogamy of the Gentoo penguin). The preliminary results showed that these nested primers could also be used for sex identification of Chinstrap penguins (Pygoscelis antarctica) and King penguins (Aptenodytes patagonicus). In conclusion, we successfully sexed Gentoo penguins using DNA extracted from fecal samples and performed nested PCR of sex-specific CHD genes.

A reliable non-invasive PCR method for takin (Budorcas taxicolor) sex identification based on amelogenin gene

We developed a reliable non-invasive PCR method for sex identification of the takin (Budorcas taxicolor) based on Amelogenin (AMEL) genes. In takin, a 45 bp deletion of Y-linked allele provides a significant distinction between AMELX and AMELY, and amplification products show sex-specific banding patterns (male: 240 and 195 bp; female: 240 bp) after agarose gel electrophoresis. However, SRY gene produces only male-specific amplification, and amplification products have only one band (male: 230 bp). Both feces and muscle samples from known-sex takins were successfully amplified. There was no amplification failure of our specific primers, and the phenotypic and genotypic of 15 tested taxins (4 males and 11 females) were highly consistent. Cross-species detection also proved that our primers could be applied to other Caprinae species. The detection sensitivity of this method was 50 pg of genomic DNA. These results show that using one pair of primers to co-amplify homologous fragments according to the AMEL gene exon 5 is a reliable and rapid method.

Coincident mass extirpation of neotropical amphibians with the emergence of the infectious fungal pathogen Batrachochytrium dendrobatidis

Amphibians highlight the global biodiversity crisis because ∼40% of all amphibian species are currently in decline. Species have disappeared even in protected habitats (e.g., the enigmatic extinction of the golden toad, Bufo periglenes, from Costa Rica). The emergence of a fungal pathogen, Batrachochytrium dendrobatidis (Bd), has been implicated in a number of declines that have occurred in the last decade, but few studies have been able to test retroactively whether Bd emergence was linked to earlier declines and extinctions. We describe a noninvasive PCR sampling technique that detects Bd in formalin-preserved museum specimens. We detected Bd by PCR in 83-90% (n = 38) of samples that were identified as positive by histology. We examined specimens collected before, during, and after major amphibian decline events at established study sites in southern Mexico, Guatemala, and Costa Rica. A pattern of Bd emergence coincident with decline at these localities is revealed-the absence of Bd over multiple years at all localities followed by the concurrent emergence of Bd in various species at each locality during a period of population decline. The geographical and chronological emergence of Bd at these localities also indicates a southbound spread from southern Mexico in the early 1970s to western Guatemala in the 1980s/1990s and to Monteverde, Costa Rica by 1987. We find evidence of a historical "Bd epidemic wave" that began in Mexico and subsequently spread to Central America. We describe a technique that can be used to screen museum specimens from other amphibian decline sites around the world.

Noninvasive Molecular Diagnosis of Human Visceral Leishmaniasis

Previously developed methods for noninvasive PCR diagnosis of visceral leishmaniasis (VL) have significant limitations. Diagnosis of VL using PCR and buccal swabs was evaluated in 307 subjects, including 148 patients confirmed to have VL. This method is simple and well tolerated and has good potential for development, showing 83% sensitivity with 90.56% specificity in control groups.

Fast and non-invasive PCR sexing of primates: apes, Old World monkeys, New World monkeys and Strepsirrhines

BackgroundOne of the key tools for determining the social structure of wild and endangered primates is the ability to sex DNA from small amounts of non-invasive samples that are likely to include highly degraded DNA. Traditional markers for molecular sex determination of primates are developed on the basis of the human sequence and are often non-functional in distantly related primate species. Hence, it is highly desirable to develop markers that simultaneously detect Y- and X-chromosome specific sequences and also work across many species.ResultsA novel method for sex identification in primates is described using a triple primer PCR reaction and agarose gel electrophoresis of the sex-chromosomal isoforms of the ubiquitously transcribed tetratricopeptide repeat protein gene (UTX/UTY). By comparing genomic data from several mammals we identified the UTX/UTY locus as the best candidate for a universal primate sexing marker. Using data from several species we identified a XY-conserved region, a Y conserved region and an X conserved region. This enabled the design of a triple primer PCR setup that amplifies X and Y products of different length in a single PCR reaction.ConclusionThis simple PCR amplification of X and Y fragments is useful for sexing DNA samples from all species of primates. Furthermore, since the amplified fragments are very short the method can be applied to fragmented DNA extracted from non-invasive samples.

Rh proteins: Key structural and functional components of the red cell membrane

Rh (Rhesus) proteins (D, CcEe) are expressed in red cells (RBC) in association with other membrane proteins (RhAG, LW, CD47 and GPB). By interacting with the spectrin-based skeleton through protein 4.2 and ankyrin, the Rh complex contributes to the maintenance of the mechanical properties of the erythrocyte membrane. The RH system is one of the most immunogenic and polymorphic human blood group system. Molecular basis of most Rh phenotypes, including the Rh null phenotype associated with hemolytic anemia, have been determined. The demonstration that the RHD -positive locus is composed of the RHD and RHCE genes, whereas the RHD gene is deleted in most RhD-negative individuals, allowed fetal RhD genotyping by non-invasive PCR assays for antenatal diagnosis of pregnancy at risk for Rh hemolytic disease of the newborn. In mammals, the Rh protein family includes two non-erythroid members, RhBG and RhCG, mainly expressed in liver and kidney, two organs specialized in ammonia genesis and excretion. Functional analyses in heterologous systems revealed that RhAG, RhBG and RhCG can mediate ammonium (NH 3 and/or NH 4 + ) transport across the cell membrane and might represent mammalian specific ammonium transporters. Furthermore, recent studies performed in human and murine red blood cells (RBC) indicate that RhAG facilitates CH 3NH 2/NH 3 movement across the membrane and represents a potential example of gas channel. The crystallographic structure of the bacterial ammonia channel AmtB and functional studies showing that AmtB conducts NH 3 into reconstituted vesicles is fully consistent with these latter studies. In RBCs , RhAG may transport NH 3 to detoxifying organs like kidney and liver and with non-erythroid tissues orthologs may contribute to regulation of the acid-base balance.

Accurate Diagnosis of Helicobacter Pylori: Polymerase Chain Reaction Tests

Although PCR has improved considerably the sensitivity of the diagnosis of H. pylori infection, many studies have not shown conclusively the full potential of PCR in clinical diagnosis. In daily clinical practice, PCR does not have to be performed to establish H. pylori infection. PCR is still classified primarily as a research technique in the Helicobacter field. PCR or similar technology will expand in the future when automation and commercialized kits are available to most laboratories. The development of a noninvasive PCR test may prove useful because it may lead ultimately to the determination of the source and route of transmission of this important pathogen.

Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri.

A plasmid of about 140 megadaltons has been associated with the invasiveness of Shigella flexneri. Upon subculturing in liquid media of fully virulent isolates of Shigella flexneri 2a YSH6000, which contains only a 230-kilobase-pair (kbp) plasmid in addition to 3.3- and 4.2-kbp cryptic plasmids characteristic to all S. flexneri strains, loss of invasiveness, loss of Congo red binding activity (Pcr), and complete loss of, or a deletion, or even a single-site IS insertion in the plasmid occurred simultaneously. This was ascribed to the fact that, once a noninvasive Pcr- cell has emerged, it overgrows the wild type as a consequence of its selective advantage in artificial media. A deletion map of the 230-kbp plasmid was made by analyzing SalI digests of 39 deletion derivatives plus 1 formed by insertion of an IS1-like element in independently isolated, noninvasive Pcr- mutants. Of 39 deletion derivatives, 16 belonged to a single type, and 6 belonged to another, suggesting deletion hot spots. The deletion map was confirmed and extended by analyzing 359 SalI-generated partial digests of the wild-type plasmid cloned into pBR322. Three copies of IS1-like elements were found on three different SalI fragments by Southern hybridization. Segments required for the Pcr+ phenotype seemed to occur at several different locations in the plasmid. Each of 28 representative Pcr- mutants were negative by the Sereny test. Hence, many, or possibly all, Pcr determinants were required for full virulence.