All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of bp in gel electrophoresis. No such. Page 1. Page 2. Page 3. Page 4. Jul 9, alternative to cytogenetics and slower PCR assays. Acute promyelocytic Am J Clin Pathol ; © American Society of Clinical.
|Language:||English, Spanish, Dutch|
|Genre:||Health & Fitness|
|ePub File Size:||25.65 MB|
|PDF File Size:||8.14 MB|
|Distribution:||Free* [*Regsitration Required]|
PCR Course Guide ii. COURSE. GUIDE. PCR DEMOCRACY AND GOOD GOVERNANCE. Course Developer. Dr. C.N. Odock,. University of. Quantification strategies in real-time PCR Quantification strategies in real- time PCR. Michael W. Pfaffl. Chaper 3 pages 87 - in: A-Z of quantitative PCR . The efficacy of the antigen-capture PCR (AC-PCR) method for the detection of in the UK by the polymerase chain reaction. FEMS. Microbiol. Lett.
Nevertheless, no association of APOE genotypes with schizophrenia was observed in this study. Twenty-three of 6. Acknowledgments The author wants to thank Mr. Bertrand, L. Specificity test of simplex PCR showed that three different-sized amplicons were amplified: Samples G. Although trichomonas PCR requires more technical skill, molecular amplification techniques are currently in use in many laboratories for the detection of C.
April 14, This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
APOE gene encodes the apolipoprotein E, which is involved in the transportation of cholesterol and lipids in blood circulation and central nervous system. Apolipoprotein E also mediates lipoprotein internalization and degradation via receptor-mediated endocytosis pathway and plays a crucial role in lipid homeostasis.
There are three major isoforms of APOE gene, E2, E3, and E4, which are determined by the genetic polymorphisms at codon rs and codon rs There are functional differences in their binding ability to lipids and receptors among different isoforms of apolipoprotein E [ 1 — 3 ].
These genetic polymorphisms of APOE gene are associated with cardiovascular diseases and neurodegenerative diseases [ 3 , 4 ].
With the expanding knowledge of the function of apolipoprotein, APOE has also been implicated in the neurobiology of psychiatric disorders [ 8 , 9 ]. Genetic association studies of APOE variants have been conducted in various psychiatric illnesses, such as major depressive disorder [ 10 , 11 ], schizophrenia [ 12 — 14 ], bipolar disorder [ 15 — 17 ], autism [ 18 — 20 ], attention deficit hyperactivity disorder [ 21 ], panic disorder [ 22 ], and post-traumatic stress disorder [ 23 , 24 ].
In addition to the association with disease risk, genetic polymorphisms of APOE were also associated with efficacy and adverse effects of certain drugs, outcome and prognosis of some diseases [ 3 , 25 , 26 ]. Hence, genotyping of APOE has broad applications in biomedical research, particularly in the era of precision medicine [ 3 ]. These methods have different requirements, advantages, and limitations.
Hence, there is a need for a convenient method that can be quickly adopted in various laboratories. We demonstrated that this method is fast, accurate, and robust and has a flexible throughput that can be used efficiently in different research and clinical laboratories. DNA samples used in this study were taken from our previous collection for series of the molecular genetic study of schizophrenia. All the subjects were Han Chinese from Taiwan. This study comprised DNA samples of patients with schizophrenia and control subjects collected from for our serial genetic study of schizophrenia.
The study was approved by the Medical Research Committee of National Health Research Institutes of Taiwan, and written informed consent was obtained from each participant and their guardians after the whole procedures were fully explained. To verify the accuracy of the melting curve-based allele-specific PCR for APOE genotyping, DNA samples selected at random from patients with schizophrenia and control subjects were genotyped using the melting curve-based allele-specific PCR method and the Sanger sequencing method simultaneously.
The genotyping results were compared between the two methods. After validation of the melting curve-based allele-specific PCR for APOE genotyping, we applied this method to conduct a genetic association study of APOE with schizophrenia consisting of patients with schizophrenia and control subjects. Differences in the genotype and allele frequency between the two groups were assessed using a chi-squared test. A P value less than 0.
A 3 mm diameter disc was punched from each Guthrie card and collected in the PCR tube. Each disc was immersed in a 50 microliter mixture of methanol and acetone 1: After drying, the disc was incubated in 50 microliter water at room temperature for at least 2 hours.
We further tested whether the whole blood lysate without DNA purification can be directly used in the melting curve-based allele-specific PCR method. After thawing and vortexing, the mixtures were centrifuged briefly. The bp amplicons were subsequently subjected to APOE genotyping using the melting curve-based allele-specific PCR method and Sanger sequencing method as described. The representative results of the melting curve-based allele-specific PCR genotyping of the codon rs using purified genomic DNA are shown in Fig 1A.
The TT homozygote had a distinct melting temperature from the CC homozygote while the TC heterozygote had two melting temperatures corresponding to that of TT and CC homozygote, respectively. Fig 1B demonstrates the representative results of the melting curve-based genotyping of the codon rs After genotyping a total of DNA samples using both the melting curve-based allele-specific PCR method and the Sanger sequencing genotyping method simultaneously.
Representative sequencing results of are shown in Fig 2. The arrow indicates the position of the single nucleotide polymorphism. B PCR amplicon generated from Guthrie blood spot. C PCR amplicon generated from a crude lysate of peripheral blood cells C. The melting curve-based allele-specific PCR method was applied to a genetic association study of APOE and schizophrenia in a sample of patients with schizophrenia and control subjects from Taiwan.
The genotype and allele frequencies of these subjects are listed in Table 2. No significant differences in the genotype and allele frequencies were detected between these two groups. A total of 10 Guthrie cards containing blood spots collected from 10 individuals were tested in this study. The storage duration of these cards ranged from 18—20 years. We were able to determine the APOE genotype of these ten individuals using this method data not shown. The authenticity of the genotype of these ten individuals was verified using Sanger sequencing method that used aliquots of the same DNA solution collected from Guthrie card.
A total of 10 samples collected from 10 individuals were tested in this study.
As shown in Fig 3C , the bp PCR products were successfully obtained from aliquots 2 microliter of crude blood cell lysate.
Aliquots of the amplicons were further subjected to APOE genotyping using the melting curve-based allele-specific PCR method and the Sanger sequencing method successfully data not shown. In this method, two allele-specific forward PCR primers were tagged with two different lengths of GC tail, respectively, which generated two allele-specific PCR amplicons with different sizes.
As melting temperature of PCR amplicon is size dependent, the two different sizes of allele-specific amplicons result in two distinct melting temperatures in dissociation curve analysis. Our method used dissociation curve analysis of default setting of the machine and did not need to use dedicated software. Hence, the protocol should be easily adopted in other machines.
The success of our protocol also lends further support to the general utility of the Tm-shift SNP genotyping method in selective SNPs of interest [ 30 ]. Nevertheless, as shown in Fig 1 , there is a high baseline before the peak of melt temperature, which might be due to the presence of some non-specific PCR products. But our method is faster and more cost savings than the Sanger sequencing method. Furthermore, when our method was applied to the genetic association study of APOE polymorphisms and schizophrenia in our population, the genotyping of patients with schizophrenia and healthy control subjects was completed in one month by one research assistant, suggesting this is an efficient APOE genotyping method.
Nevertheless, no association of APOE genotypes with schizophrenia was observed in this study. Several case-control studies have been conducted on the genetic association of APOE and schizophrenia. The results have been inconsistent and equivocal in different studies [ 12 , 31 ].
In a meta-analysis of the genetic association of APOE with schizophrenia that consisted of 11 Caucasian and 6 Asian case-control studies, APOE4 was found to have a modest association of risk with schizophrenia in the Caucasian population, but not in Asian population [ 12 ].
Furthermore, no other APOE alleles were found to be associated with schizophrenia in this meta-analysis in either Caucasian or Asian samples when analyzed separately or together [ 12 ]. The other alleles were not found to have a significant association in their study [ 31 ]. We did not observe the protective effective of APOE3 against schizophrenia in our present study, nor did we observe the association of other alleles of APOE with schizophrenia.
Nevertheless, the genetic data of our study shall contribute to future meta-analysis study of APOE and schizophrenia. Also, our genetic data would be useful for the case-control association study of APOE genotype with other diseases such as type 2 diabetes and coronary artery disease in our population [ 32 ]. In this study, we also demonstrated that blood samples collected on Guthrie blood filter paper that were used in the screening program of congenital errors of metabolism can be used directly in the melting curve-based allele-specific PCR APOE genotyping assay.
Thus, preventive procedures may be adopted to avoid or delay the onset of these diseases in their later life. The causes might be due to some materials present in the crude blood cell lysate that may inhibit PCR and interfere with the fluorescence detection. Further troubleshooting is needed to overcome this issue.
Thus, although indirectly, the use of crude blood cell lysate in the melting curve-based allele-specific PCR APOE genotyping represents a fast and cost-saving method that does not need DNA purification from blood cells. Hence, this is a convenient method and cost saving method that can be used in a large-scale population study. In summary, we developed a melting curve-based allele-specific PCR method for APOE genotyping that is efficient, convenient, and accurate and has the flexibility to suit different sample size.
The method is versatile and robust that different preparations of DNA can be used in this method. Thus, the method should potentially have a broad range of applications in clinical and research laboratories.
Conceived and designed the experiments: Performed the experiments: Analyzed the data: All three targeted bands were amplified in one tube simultaneously Figure 1 , and G. Optimal amplification results were obtained with 1.
The sensitivity test revealed that the detection limit was 10 eggs for A. Of fecal samples, Data analysis showed that the prevalence of A. In addition, there were no significant differences in the prevalence of A. It is known that A.
In addition to the veterinary importance, those parasites can also cause zoonotic diseases in human. To date, many methods have been developed for the identification and detection of those parasites, such as the high-resolution melting HRM assay established by Zhang et al. However, prior to the present study, there had been no report on the utilization of multiplex PCR approaches for detection of hookworm and G.
This work describes the development of a multiplex PCR method for simultaneous detection of A. In the process of establishing multiple PCR detection methods, the primer design is a key point. Firstly, the selected primer design region must be highly specific to avoid competitive amplification among target fragments.
In addition, annealing temperature of those primers was relatively close, making successful amplification of all fragments under the same temperature. Regarding specificity, this method was specific to amplify the three target fragments, and no cross-reaction with the common intestinal parasites, such as Toxocara cati , Trichuris vulpis , Dipylidium caninum , Isospora felis , and Giardia lamblia assemblages C, D, and F, of dogs and cats was observed.
As for sensitivity test of A. Unfortunately, the small size of G.
Our results showed that the detection limit was 10 eggs for A. By using the established multiplex PCR detection method in present study, Thus it supports the correctness of our previous study, or vice versa, as well as the suggestion that the predominant species of hookworms in cats was A.
In addition, 2.
Even though it was lower compared with the reported value of 8. In conclusion, this work presents the first report on the development of a multiplex PCR for simultaneous detection of three important zoonotic parasites: The results indicate that this method was a rapid and effective method for the identification and detection of those zoonotic parasites. The clinical detection results are in line with those previously detected by PCR-RFLP method and show that the cats in Guangzhou harbor all these three zoonotic parasites.
Briefly, humans can become infected by these three zoonotic parasites through oral ingestion and percutaneous migration of infective L3 larvae A. Therefore, control measures are recommended to prevent and reduce the infection risk from cats to humans.
The authors declare that there is no conflict of interests regarding the publication of this paper. BioMed Research International. Indexed in Science Citation Index Expanded. Journal Menu. Special Issues Menu. Subscribe to Table of Contents Alerts. Table of Contents Alerts. Abstract Ancylostoma ceylanicum , A.
Introduction Hookworms are blood-feeding parasitic intestinal nematodes which infect humans, dogs, cats, and other mammals throughout the world. Materials and Methods 2. Samples G.
Table 1: Primers for the simultaneous detection of Ancylostoma ceylanicum , A. Figure 1: Interaction effect of multiplex PCR primers.
Figure 2: Optimization of Giardia lamblia assemblage A primers concentration. Figure 3: Optimization of annealing temperature. Figure 4: Figure 5: Specificity of multiplex PCR. Toxocara cati ; 9: Trichuris vulpis ; Dipylidium caninum ; Isospora felis ; Sterile ddH 2 O. Figure 6: Sensitivity of multiplex PCR. Table 3: References D. Bowman, S. Montgomery, A. Zajac, M. Eberhard, and K. Traub, T. Inpankaew, C. Sutthikornchai, Y. Sukthana, and R. Jiraanankul, W. Aphijirawat, M. Mungthin et al.
Ngui, Y. Lim, R. Traub, R. Mahmud, and M.
Conlan, B. Khamlome, K. Vongxay et al. Inpankaew, F. Dalsgaard et al. Landmann and P. Feng and L. Zheng, M. Alsarakibi, Y. Liu et al. Zheng, W. Hu, Y. Liu, Q.