Polymerase Chain Reaction Capillary Electrophoresis Research Articles

Assessing T-cell receptor clonality by next-generation sequencing in atypical cutaneous lymphoid infiltrates and cutaneous T-cell lymphoma: A scoping review.

The diagnosis of cutaneous T-cell lymphoma (CTCL) remains challenging. Demonstration of a clonal T-cell population using T-cell receptor (TCR) gene rearrangement studies by next-generation sequencing (NGS) has been explored in several studies. This review summarizes the current literature on NGS-based sequencing methods for the assessment of TCR clonality in the evaluation of atypical cutaneous lymphoid infiltrates and CTCL on behalf of the American Society of Dermatopathology Appropriate Use Criteria Committee (lymphoproliferative subgroup). PubMed was searched for relevant articles, including CTCL and NGS, for clonality from 1967 to 2022. Thirteen studies were included in the analysis. The skin was the most commonly assayed compartment with TCR NGS. Sensitivity for TCR NGS in the skin ranged between 69% and 100%, compared to 44%-72% for polymerase chain reaction (PCR)-capillary electrophoresis. Specificity for TCR NGS in the skin ranged from 86% to 100%, compared to 77%-88% for PCR capillary electrophoresis. TCR NGS was also reported to have potential prognostic value in CTCL and can also be used to detect relapse and/or minimal residual disease after treatment.

Clinical Value of Different Test Methods in Diagnosing Mycoplasma pneumoniae Infection in Children.

This research aimed to investigate the diagnostic value of passive particle agglutination test, Mycoplasma pneumoniae (MP) culture, cold agglutination test (CAT), enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction-capillary electrophoresis fragment analysis (PCR-CEFA) for MP infection. Children with respiratory tract infections suspected to be MP infection were subjected to passive particle agglutination test, MP culture, CAT, ELISA, and PCR-CEFA. A total of 146 children (81 males, 65 females, mean age: 5.74 ± 3.32 years, and mean course of disease: 9.07 ± 5.18 days) met the inclusion criteria. The positivity rate of MP detection by MP culture was 69.18% (101/146). Using the MP culture method as the standard, higher sensitivity and positive predictive value were found in the PCR-CEFA compared with the other 3 methods. Appropriate methods are selected following the advantages and disadvantages of pathogen detection, and pediatric MP infection is analyzed by integrating various test results.

Impact of Micronutrient Profiles on the Pathological Features and Clinical Outcomes of Colorectal Tumors with Microsatellite Instability

We assessed the role of nutritional factors in conjunction with pathological and clinical characteristics of colorectal tumors with microsatellite instability. Data from 1,135 patients diagnosed with colorectal cancer were analyzed, employing immunohistochemistry for mismatch repair protein detection and polymerase chain reaction-capillary electrophoresis for microsatellite instability gene detection. To explore potential correlations with microsatellite instability status, we evaluated patients’ dietary habits and micronutrient levels, particularly folate and selenium. We discovered that 10.84% of the patients had mismatch repair protein deletions, with MLH1 and PMS2 double deletions being the most prevalent. Notable differences were identified in serum carcinoembryonic antigen levels, tumor location, tumor-node-metastasis stage, tumor differentiation, mucous composition, gross type, size, nodules, nerve invasion, and lymph node ratio between deficient mismatch repair and proficient mismatch repair patients. The deficient mismatch repair group had lower carcinoembryonic antigen levels, a higher incidence of right-sided colon cancer, earlier tumor-node-metastasis stage, poorer differentiation, more mucinous components, larger tumors, and fewer nodules and nerve invasions. High concordance (98.59%) was observed between immunohistochemistry and polymerase chain reaction-capillary electrophoresis techniques in microsatellite instability detection, underscoring the reliability of these methods. Integrating the nutritional parameters with conventional clinical assessments offers a nuanced understanding of the heterogeneity of colorectal cancer, with significant implications for diagnosis and treatment strategies.

Short tandem repeat profiling via next-generation sequencing for cell line authentication.

Cell lines are indispensable models for modern biomedical research. A large part of their usefulness derives from the ability of a cell line to proliferate over multiple passages (often indefinitely), allowing multiple experiments to be performed. However, over time, cell line identity and purity can be compromised by human errors. Cross-contamination from other cell lines and complete misidentification are both possible. Routine cell line authentication is a necessary preventive measure and has become a requirement for many funding applications and publications. Short tandem repeat (STR) profiling is the most common method for cell line authentication and is usually carried out using standard polymerase chain reaction-capillary electrophoresis analysis (STR-CE). Here, we evaluated next-generation sequencing (NGS)-based STR profiling of human and mouse cell lines at 18 and 15 loci, respectively, in a high-throughput format. Using the Python program STRight, we demonstrate that NGS-based analysis (STR-NGS) is superior to standard STR-CE in terms of the ability to report the sequence context of repeat motifs, sensitivity and flexible multiplexing capability. STR-NGS is thus a valuable alternative for cell line authentication.

A retrospective study of consistency between immunohistochemistry and polymerase chain reaction of microsatellite instability in endometrial cancer.

Identification of endometrial cancers (EC) with mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H) is essential for Lynch syndrome screening and treatment stratification. We aimed to assess the utility of immunohistochemistry (IHC) staining for MMR protein expression and polymerase chain reaction (PCR)-based MSI assays in EC and the correlation between MMR/MSI status and various clinicopathological parameters. We reviewed the clinical and pathological information of 333 patients with EC. MMR protein expression was assessed as retained or lost to determine MMR status by IHC staining, and MSI status was identified by PCR capillary electrophoresis (PCR-CE) testing with a National Cancer Institute (NCI) panel. The correlation of MMR/MSI status with clinicopathological features was determined by statistical analysis. Discrepant results were further analyzed using an alternative PCR-CE MSI (Promega panel) method, MLH1 promoter methylation assays, and next-generation sequencing (NGS). Among the EC patients, the overall percentage of dMMR was 25.2%, and the overall percentage of MSI-H was 24%. Among the dMMR patients, 50 (59.5%) showed loss of MLH1 and PMS2 expression, 19 (22.6%) loss of MSH2 and MSH6 expression, and seven (8.3%) and eight (9.5%) loss of PMS2 and MSH6 expression, respectively. The dMMR subgroup was significantly younger than the pMMR subgroup, especially for <60-years-old patients (p=0.038). In addition, we identified a strong correlation between MMR/MSI status and high-grade endometrioid or nonendometrioid components (p=0.004 or p=0.003). IHC staining and PCR-CE assay results showed a high level of overall concordance (98.8%, Cohen's κ=0.98). Four patients were found to have dMRR/MSS in both examinations. We reanalyzed them with additional methods. One case showed MLH1 promotor methylation, and the other three cases harbored MSH6 germline pathogenic variations. One of the cases with MSH6 deficiency was reanalyzed as MSI-H by alternative PCR-CE assay or NGS testing. This study indicates that the combined use of MMR-IHC and PCR-CE MSI analyses may effectively avoid misdiagnoses of EC patients with dMMR/MSI-H. However, use of PCR-CE alone to evaluate MMR/MSI status may lead to missed diagnosis, especially for EC patients with MSH6 deficiency and presenting MSS.

Heterogeneous expression of mismatch repair proteins and interpretation of immunohistochemical results in colorectal cancer and endometrial cancer

To investigate the heterogeneous expression patterns of four mismatch repair (MMR) proteins in colorectal cancer (CRC) and endometrial cancer (EC), and their effects on the interpretation of immunohistochemical (IHC) results. A total of 1636 CRC and EC specimens were collected from two hospitals. Seventy-eight cases had heterogeneous expression of MMR proteins, including 49 CRC and 29 EC cases. Polymerase chain reaction-capillary electrophoresis (PCR-CE) was then performed to detect the microsatellite instability (MSI) status, and 44 cases were further verified by targeted next-generation sequencing (NGS). Heterogeneous expression of MMR proteins was observed in 66 cases (66/78, 84.6%) of proficient MMR (pMMR) and 12 cases (12/78, 15.4%) of deficient MMR (dMMR). The proportion of heterogeneous MMR protein expression in EC (12.0%) was higher than that in CRC (3.5%). The heterogeneous expression patterns were divided into focal clonal heterogeneity (6/78, 7.7%) and glandular mosaic heterogeneity (72/78, 92.3%). Surprisingly, three pMMR CRC cases showed isolated small focal clonal heterogeneity of mutS homologue 6 (MSH6), with < 15% positive tumour cells, which was validated as high MSI (MSI-H) with PCR-CE and NGS. However, the other three EC pMMR cases with > 50% focal clonal heterogeneity of MMR proteins were verified as microsatellite stable (MSS) or low MSI (MSI-L). Fifteen EC cases with glandular mosaic heterogeneous expression of MMR proteins included two MSI-H cases, which were validated using PCR-CE and NGS. Among the dMMR cases, only two EC cases with mutL homologue 1 (MLH1)/PMS1 homologue 2, mismatch repair system component (PMS2) loss and MSH2/MSH6 mosaic heterogeneous expression were confirmed as MSS using PCR-CE and NGS, which may be related to the mechanism of MLH1 promoter methylation. Thus, in CRC, only cases with small focal clonal heterogeneous expression of MSH6 have a high likelihood of MSI-H, and further PCR-CE or NGS testing is recommended. The possibility of MSI-H cannot be ruled out in EC cases with glandular mosaic heterogeneous expression of MMR proteins; PCR-CE or NGS detection is therefore necessary.

Concordance between microsatellite instability and mismatch repair protein expression in colorectal cancer and their clinicopathological characteristics: a retrospective analysis of 502 cases.

Microsatellite instability (MSI) is one of the hallmarks of colorectal cancer (CRC). Mismatch repair (MMR) protein expression may reflect MSI status. To analyze the concordance between MSI and MMR expression in CRC and their clinicopathological characteristics, 502 CRC patients were retrospectively collected in this study. Polymerase chain reaction-capillary electrophoresis (PCR-CE) was used to measure MSI, and MMR expression was determined by immunohistochemistry (IHC). The causes of non-concordance were analyzed. Chi-square test was used to find the correlation between MSI and various clinicopathological parameters. PCR-CE results showed 64 (12.7%) patients had high microsatellite instability (MSI-H); low microsatellite instability (MSI-L) and microsatellite stable (MSS) cases were 19 (3.8%)and 419 (83.5%), respectively. With regard to IHC, 430 (85.7%) showed proficient mismatch repair (pMMR) and 72 (14.3%) showed deficient mismatch repair (dMMR). The coincidence rate of MSI and MMR expression in CRC was 98.4% (494/502), with good concordance (Kappa = 0.932). Using PCR-CE as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of IHC were 100%, 98.2%, 88.9%, and 100%, respectively. MSI-H was more common in women, right colon, tumors ≥ 5cm, ulcerative type, mucinous adenocarcinoma, poor differentiation, T stage I/II, and without lymph node or distant metastasis for CRC patients. In summary, MSI exhibited some typical clinicopathological characteristics. MSI and MMR expression in CRC had good concordance. However, it is still extremely necessary to perform PCR-CE. We recommend that testing packages of different sizes should be developed in clinical practice to create a testing echelon, to facilitate comprehensive selection according to experimental conditions, clinical diagnosis, and treatment needs.

Forensic Validation Studies of a Novel 35-InDel Multiplex Polymerase Chain Reaction System

Abstract Background: A difficulty associated with forensic applications is the detection of degraded biological materials. Due to the large amplicon sizes of short tandem repeat alleles, valid genotyping results cannot be obtained from degraded biological materials. Recently, insertion/deletion (InDel) polymorphisms have been used in forensic applications for their widespread distributions in the human genome, short amplicon sizes, and low mutation rates. Purpose: Human identification InDel panels have mostly been designed for European populations. Therefore, our laboratory independently developed a multiplex polymerase chain reaction (PCR) system with 35 polymorphic InDel loci to be used for human identification in China. Forensic validation studies were conducted on this novel 35-InDel multiplex PCR system. Methods: The 35 InDel loci were screened in the database, and then used with the traditional PCR amplification and capillary electrophoresis platform combined with five-color fluorescence parallel detection technology. Validation studies were performed on this novel panel, including accuracy, repeatability and reproducibility, species specificity, sensitivity, stability, forensic case sample detection, and mixture studies. In addition, forensic efficiency assessments were conducted in populations from different continents. Results: The data of validated studies indicated that the novel 35-InDel panel was accurate, stable, and efficient for forensic purposes. For human identification, the cumulative power of discrimination values for the these 35 InDel loci in East Asian, South Asian, European, American, and African populations were 0.999999999999995, 0.999999999999995, 0.999999999999971, 0.9999999999999960, and 0.999999999998166, respectively. Conclusions: In this study, a set of 35 InDel loci were conducted in a multiplex amplification system for human identification of degraded DNA sample, and this new assay was efficient and stable. The present results suggested that the 35-InDel panel was a reliable tool for forensic use and could be efficiently used for human identification in the East Asian populations.

Combining Direct PCR Technology and Capillary Electrophoresis for an Easy-to-Operate and Highly Sensitive Infectious Disease Detection System for Shrimp.

Infectious diseases are considered the greatest threat to the modern high-density shrimp aquaculture industry. Specificity, rapidity, and sensitivity of molecular diagnostic methods for the detection of asymptomatic infected shrimp allows preventive measures to be taken before disease outbreaks. Routine molecular detection of pathogens in infected shrimp can be made easier with the use of a direct polymerase chain reaction (PCR). In this study, four direct PCR reagent brands were tested, and results showed that the detection signal of direct PCR in hepatopancreatic tissue was more severely affected. In addition, portable capillary electrophoresis was applied to improve sensitivity and specificity, resulting in a pathogen detection limit of 25 copies/PCR-reaction. Juvenile shrimp from five different aquaculture ponds were tested for white spot syndrome virus infection, and the results were consistent with the Organization for Animal Health’s certified standard method. Furthermore, this methodology could be used to examine single post larvae shrimp. The overall detection time was reduced by more than 58.2%. Therefore, the combination of direct PCR and capillary electrophoresis for on-site examination is valuable and has potential as a suitable tool for diagnostic, epidemiological, and pathological studies of shrimp aquaculture.

Development of SSR Databases Available for Both NGS and Capillary Electrophoresis in Apple, Pear and Tea.

Developing new varieties in fruit and tea breeding programs is very costly and labor-intensive. Thus, establishing a variety discrimination system is important for protecting breeders’ rights and producers’ profits. Simple sequence repeat (SSR) databases that can be utilized for both next-generation sequencing (SSR-GBS) and polymerase chain reaction–capillary electrophoresis (PCR-CE) would be very useful in variety discrimination. In the present study, SSRs with tri-, tetra- and pentanucleotide repeats were examined in apple, pear and tea. Out of 37 SSRs that showed clear results in PCR-CE, 27 were suitable for SSR-GBS. Among the remaining markers, there was allele dropout for some markers that caused differences between the results of PCR-CE and SSR-GBS. For the selected 27 markers, the alleles detected by SSR-GBS were comparable to those detected by PCR-CE. Furthermore, we developed a computational pipeline for automated genotyping using SSR-GBS by setting a value “α” for each marker, a criterion whether a genotype is homozygous or heterozygous based on allele frequency. The set of 27 markers contains 10, 8 and 9 SSRs for apple, pear and tea, respectively, that are useful for both PCR-CE and SSR-GBS and suitable for automation. The databases help researchers discriminate varieties in various ways depending on sample size, markers and methods.

Evaluating the effects of whole genome amplification strategies for amplifying trace DNA using capillary electrophoresis and massive parallel sequencing

To draw robust conclusions when trace DNA samples are detected in complex cases, it is essential to successfully recover and genotype short tandem repeats (STRs) from trace DNA. However, obtaining complete STR profiles by the conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) method is generally difficult as trace DNA is often less than 100 pg. Previous studies have proven that through whole-genome amplification (WGA), the yield of DNA from trace DNA samples could be improved. In this study, we used two WGA kits, namely, REPLI-g® Single Cell kit and MALBAC® Single Cell DNA Quick-Amp Kit (hereafter referred to as REPLI and MALBAC), to amplify DNA samples with a series of dilutions (from 5.00 ng/μL to 0.391 pg/μL). Typing of STR markers in samples with and without WGA were then performed on a CE platform by the application of Goldeneye® DNA ID System 20 A kit, as well as directly calling sequences from massive parallel sequencing (MPS) for WGA samples with 1.00 ng, 125 pg and 25.0 pg as DNA inputs. Quantification results demonstrated that the yield of samples with WGA could reach the microgram level. The amplification fold was at least > 2000 and > 200 for REPLI and MALBAC, respectively. CE results showed that the number of correctly called loci was improved for trace DNA after WGA when the DNA inputs were lower than 25.0 pg for REPLI and 6.25 pg for MALBAC, respectively. WGA remarkably improved the percentage of called loci with DNA inputs lower than 50.0 pg, although poor performance in repeatability was observed. MPS results suggested that the correctly called loci calculated by MPS reads were mostly more than those calculated by CE, particularly for those of short length, implying MPS of samples after WGA is worth testing in the future. In conclusion, WGA has the potential usability for forensic trace DNA analysis at the single-cell level with good fidelity, although its repeatability requires further improvement.

Application of Chlorophyte ChlB Gene and Cyanophyte NIES Gene in the Detection of Drowning-Related Plankton.

Objective To construct a polymerase chain reaction-capillary electrophoresis （PCR-CE） detection method using ChlB gene and NIES gene, investigate the method's specificity and sensitivity, and to evaluate its application value in drowning diagnosis. Methods The specific primers ChlB and NIES were designed for the conserved sequence of chlorophyte ChlB gene and cyanophyte NIES gene in GenBank to construct PCR-CE detection method; 50 species of standard DNA samples were amplified; the sensitivity was determined by gradient concentration detection of positive standard samples; 25 actual cadaver lung tissue samples （drowned： 20, natural death： 5） were detected, and the simultaneous detection results of microwave digestion-vacuum filtration-automated scanning electron microscopy （MD-VF-Auto SEM） were simultaneously compared. Results The minimum DNA detection concentration of primers ChlB and NIES was 0.161 ng and 0.109 ng, respectively, which could specifically amplify chlorophyte （Chlorella pyrenoidosa） and cyanophyte ［Microcystis aeruginosa （producing and not producing toxin）］ widespread in water. The product fragments were 156 bp and 182 bp, respectively. The results of non-drowning tissues were negative. Conclusion This method has high sensitivity and specificity. It can be applied to the detection of plankton related to drowning and combined with MD-VF-Auto SEM method, can increase the detection range of plankton related to drowning and improve the evidence power of drowning diagnosis.

Evaluation of Concordance Between Deficient Mismatch Repair and Microsatellite Instability Testing and Their Association with Clinicopathological Features in Colorectal Cancer.

BackgroundMicrosatellite instability (MSI) is one of the most important molecular characteristics of colorectal cancer (CRC), which mainly results from defective DNA mismatch repair (MMR). This study was performed to investigate the concordance between deficient MMR and MSI testing, and to evaluate the association of these two results with clinicopathological characteristics in Chinese CRC patients.MethodsA total of 738 CRC patients were included. Tumor tissues and paired peripheral blood specimens were obtained. Screening for MMR was investigated using immunohistochemical (IHC) technique, and multiple polymerase chain reaction-capillary electrophoresis (PCR-CE) method was performed to detect the MSI status. All clinicopathological data, immunohistochemistry and microsatellite instability analyses were then statistically analyzed.ResultsOf the 738 (17.75%) CRC patients, 131 expressed as deficient mismatch repair (dMMR) status, and postmeiotic segregation increased 2 (PMS2) deficiency was the most frequent deficiency among these four MMR proteins. MSI-high (MSI-H) status occurred in 74 of the 738 (10.03%) CRC patients, 55 of whom showed instability at all six mononucleotides repeat markers. dMMR was significantly associated with MSI-H and moderate concordance was observed between IHC and PCR-CE in evaluating deficient MMR/MSI through Kappa test. Statistically, dMMR was significantly associated with younger age, right-sided colon and poor differentiation. MSI-H was associated with younger age, right-sided colon, poor differentiation, mucinous type and tumor, node, metastasis (TNM) stage II.ConclusionA moderate concordance between deficient MMR and MSI testing indicates that both IHC and PCR-CE methods should be routinely tested to provide reliable data for clinical treatment decisions.

The trnL (UAA)-trnF (GAA) intergenic spacer is a robust marker of green pea (Pisum sativum L.) adulteration in economically valuable pistachio nuts (Pistacia vera L.).

Pistachio (Pistacia vera L.) is an expensive culinary nut species; it is therefore susceptible to adulteration for economic profit. Green pea (Pisum sativum L.) kernels constitute the most common material used for adulterating chopped / ground pistachio nuts and pistachio paste. Food genomics enables the species composition of a food sample to be ascertained through DNA analysis. Accordingly, a barcode DNA genotyping approach was used to standardize a test method to identify green pea adulteration in pistachio nuts. The trnL (UAA)-trnF (GAA) intergenic spacer in the plastid genome was the target analyte in the present study. The barcode locus displayed a significant, discriminatory size difference between pistachio and pea, with amplicon sizes of 449 and 179 bp, respectively. Polymerase chain reaction-capillary electrophoresis (PCR-CE) analysis of the intergenic spacer resulted in the successful identification of species composition in the in-house admixtures, which contained 5% to 30% of green pea. The present work describes a fast and straightforward DNA test that identifies green pea adulteration in pistachio nuts without requiring a statistical data interpretation process. The plastid trnL (UAA)-trnF (GAA) intergenic spacer length widely varies among plant taxa, so the PCR-CE protocol that operates on the intergenic spacer holds the potential to reveal adulteration with a plethora of adulterants. The PCR-CE assay described in the present work can be adopted readily by food-quality laboratories in the public sector or the food industry as an easy and reliable method to analyze pistachio authenticity. © 2020 Society of Chemical Industry.

Detection and Quantification of FLT3 Internal Tandem Duplication Mutations Do Not Vary Significantly Between Whole Blood and Blast-Enriched Samples.

Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods. Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF. Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased. Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.

Identification of meat from yak and cattle using SNP markers with integrated allele-specific polymerase chain reaction–capillary electrophoresis method

Correct species identification is necessary for meat product quality assurance. Genetic markers are useful tools to accurately determine the animal source of meat products. To better protect yak meat and its products, ten single nucleotide polymorphism (SNP) loci from bovine meat-related genes and one positive control primer set were selected to discriminate meat sourced from yak or cattle. A multiplex allele-specific polymerase chain reaction and capillary electrophoresis method was employed for SNP genotyping. The samples were identified as from yak or cattle based on the number of amplicons. Yak samples generally produced one to three amplicon peaks, and the cattle samples yielded five or more peaks in the electropherogram. This method could be useful to distinguish yak meat from cattle beef, and would protect consumers and yak meat producers.

Filipino DNA variation at 12 X-chromosome short tandem repeat markers

Demands for solving complex kinship scenarios where only distant relatives are available for testing have risen in the past years. In these instances, other genetic markers such as X-chromosome short tandem repeat (X-STR) markers are employed to supplement autosomal and Y-chromosomal STR DNA typing. However, prior to use, the degree of STR polymorphism in the population requires evaluation through generation of an allele or haplotype frequency population database. This population database is also used for statistical evaluation of DNA typing results. Here, we report X-STR data from 143 unrelated Filipino male individuals who were genotyped via conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) using the 12 X-STR loci included in the Investigator® Argus X-12 kit (Qiagen) and via massively parallel sequencing (MPS) of seven X-STR loci included in the ForenSeq™ DNA Signature Prep kit of the MiSeq® FGx™ Forensic Genomics System (Illumina). Allele calls between PCR-CE and MPS systems were consistent (100% concordance) across seven overlapping X-STRs. Allele and haplotype frequencies and other parameters of forensic interest were calculated based on length (PCR-CE, 12 X-STRs) and sequence (MPS, seven X-STRs) variations observed in the population. Results of our study indicate that the 12 X-STRs in the PCR-CE system are highly informative for the Filipino population. MPS of seven X-STR loci identified 73 X-STR alleles compared with 55 X-STR alleles that were identified solely by length via PCR-CE. Of the 73 sequence-based alleles observed, six alleles have not been reported in the literature. The population data presented here may serve as a reference Philippine frequency database of X-STRs for forensic casework applications.

An integrated slidable direct polymerase chain reaction-capillary electrophoresis microdevice for rapid Y chromosome short tandem repeat analysis

We have developed an integrated slidable direct polymerase chain reaction-capillary electrophoresis (Direct PCR-CE) microdevice to analyze mini-Y chromosome short tandem repeat (mini-Y STR) typing without a DNA purification step. The Direct PCR has been recently commercialized as a simple DNA amplification technique, which does not require any sample preparation steps such as cell lysis, DNA extraction and purification for amplifying specific target genes. By implementing the advantages of Direct PCR on a microdevice, we can amplify target min-Y STR loci directly from human whole blood in a micro-reactor (2μL) and the amplicons were successively separated by micro-capillary electrophoresis. The utilization of a slidable plate enables us to manually control the fluid without use of microvalves and microtubes, and the functional units of sample injection, Direct PCR, and CE analysis were sequentially and independently performed by switching the slidable plate to each unit. On the integrated slidable Direct PCR-CE microdevice, we could complete monoplex and multiplex mini-Y STR typing directly using human whole blood within 1 h.

Identification of Vibrio cholerae serotypes in high-risk marine products with non-gel sieving capillary electrophoresis

Vibrio cholerae, a natural inhabitant of the marine environment, poses a threat to human health, and its new epidemic variants have been reported. A method of multiplex polymerase chain reaction–capillary electrophoresis–laser-induced fluorescence (PCR–CE–LIF) detection has been developed to detect and identify V. cholerae in marine products sensitively, rapidly, and reliably. Four sets of primers were selected to amplify genus-specific VCC gene, O139 serogroup-specific O139 gene, O1 serogroup-specific O1 gene, and ctxA gene associated with the CT toxin of enterotoxigenic V. cholerae. The PCR products were detected using CE–LIF with SYBR Gold serving as the DNA fluorescent dye. The parameters of PCR and the separation conditions of CE–LIF were optimized. Under the optimal conditions, V. cholerae was detected and four serotypes were identified simultaneously within 8 min. The alignment analysis showed that the PCR products had good agreement with the published sequences from GenBank, indicating that the primers selected in this study had high specificity and the PCR results were reliable. The proposed method could detect 5 to 20 cfu/ml V. cholerae. The intraday precisions of migration time and peak area of DNA marker and PCR products were in the ranges of 1.60–2.56% and 1.60–6.29%, respectively. The specificity results showed that only five standard bacteria used in this study showed the specific peaks when the target bacteria were mixed with seven other common intestinal pathogenic bacteria at the same concentration. The assay was applied to 71 high-risk marine products, and different serotypes of V. cholerae could be identified sensitively and reliably.

Integrated slidable and valveless polymerase chain reaction–capillary electrophoresis microdevice for pathogen detection

An integrated slidable and valveless polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for pathogen detection on a portable genetic analysis microsystem. The proposed microdevice does not need any microvalve or external pneumatic actuators, thereby simplifying the chip fabrication and the chip operation process. The slidable PCR–CE chip consists of three glass layers: a channel wafer that includes a sample injection and CE microchannel, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a slidable plate in which a PCR chamber was fabricated. First, the slidable PCR plate was placed in the sample injection zone to load a PCR cocktail, and then moved to the PCR zone for thermal cycling. After PCR, the slidable PCR chamber was switched to the CE region for separation, the resultant amplicons were then analyzed by a miniaturized laser-induced fluorescence detector. Non-specific DNA adsorption was prevented by treating the glass surface with hydrophobic decyltrichlorosilane, and the leakage and evaporation of the PCR cocktail during the movement and PCR could be minimized by surrounding the slidable chamber with mineral oil. The target protein A gene (101 bp) of Staphylococcus aureus (S. aureus) was detected with a limit of detection (LOD) of 6 copies, even with a background of 1000-fold excess of Escherichia coli K12 (E. Coli K12) templates. This demonstrates a highly sensitive and selective genetic analysis was achieved on our slidable and valveless PCR–CE microdevice.