PCR Capillary Electrophoresis Research Articles

Individual Identification and Assessment of Genetic Diversity Using Microsatellite Markers in Racing Pigeons Raised in Turkiye

The implementation of a swift and economical molecular genetic approach, ensuring both efficacy and cost-effectiveness and facilitating population certification, is of utmost significance for breeders and the conservation of Turkiye's native pigeon biodiversity. In this study, we aimed to examine the genetic structure of racing pigeons (Columba livia domestica) raised in Turkiye using a genetic marker panel consisting of eight short tandem repeat (STR) loci. For this purpose, DNA was isolated from the shed feathers of 216 pigeons. Genomic DNA was amplified using the multiplex allele-specific PCR and subsequent capillary electrophoresis with ABI PRISM 3130XL Genetic Analyzer. Next, PCR products were analyzed in the GeneMapper Software program (Applied Biosystems). For parent testing, paternity index (PI), combined paternity index (CPI), and cumulative probability of paternity (CPP) were calculated. Furthermore, population genetic diversity was evaluated using heterozygosity (He), polymorphism information content (PIC), and Hardy–Weinberg equilibrium (HWE) testing. Results revealed that the total number of alleles is 81 and the number of alleles per locus varies between 4 and 19. The similarity rate between parent and offspring was calculated as 99.99% and above. Since no pedigree information was given when the samples were analyzed, obtaining this similarity ratio demonstrates the reliability of the panel. He values range from 0.362 to 0.919, and the PIC values range from 0.295 to 0.909. Loci PG-1, PG-2, and PG-3 show significant genetic diversity, with moderate to high PIC values reflecting varied allele frequencies in the population. Consequently, the set of seven STR markers (+ one sex marker) can be applied to identify and confirm parentage on a regular basis, thereby facilitating efficient breeding programs and ensuring genetic diversity conservation. This panel enables efficient pedigree analysis and gender determination, optimizing cost-effectiveness. The methodology presented in this study is ideal for pedigree analysis and breed certification in the Turkish pigeon breeding industry. Consequently, we affirm that the study data carries considerable national importance.

Enhanced Commendable Sensitivity and Specificity for MSI in Colorectal Cancer by a New PCR-HRM Based 8-Loci MSI Assay.

This study evaluated the performance of the PCR-HRM assay by comparing it with immunohistochemistry (IHC) for mismatch repair (MMR) proteins and the PCR capillary electrophoresis (PCR-CE) methods. A total of 224 patients with colorectal cancer participated in the study, with nearly half having mismatch repair deficiency (dMMR) tissues and the remainder possessing pMMR tissues. There was a 97.77% concordance between the PCR-HRM assay and IHC, and a 97.56% concordance between PCR-HRM and the PCR-CE assay. In comparison with IHC for dMMR proteins, the PCR-HRM demonstrated a sensitivity of 96.36% and a specificity of 99.12%. When juxtaposed with the PCR-CE assay, its sensitivity was 98.96% and specificity stood at 96.33%. The mutations observed in the microsatellite loci were uniformly distributed across all eight loci. Discrepant outcomes were more frequent in instances of MLH1 and PMS2 deficiency. Furthermore, the germline mutation status of MLH1, MSH2, PMS2, and MSH6 in 62 patients was ascertained using next-generation sequencing. All patients displaying MMR gene pathogenic mutations (N = 14) were identified as MSI-H by PCR-HRM, whereas those with MSS tissues (N = 43) did not exhibit MMR gene pathogenic mutations. Thus, the PCR-HRM method proficiently pinpoints tumors with verified germline MMR mutations, indicative of Lynch syndrome. Conclusively, the PCR-HRM assay emerges as a swift and congruent diagnostic tool for microsatellite instability, boasting commendable sensitivity and specificity in colorectal cancer.

Assessment of Genetic Diversity and Relatedness of Local Banana (Musa spp.) Cultivars in Malaysia Using Simple Sequence Repeat (SSR) Markers

Banana (Musa spp.) is an economically important crop widely consumed all over the world. Understanding the genetic diversity and relatedness between various banana cultivars is important to continuously improve its fruit characteristics, nutritional value, and disease resistance. This study aimed to develop a DNA fingerprinting panel using polymorphic simple sequence repeat (SSR) markers for the genetic characterisation of 11 banana cultivars in Malaysia. A total of 10 polymorphic SSR markers were developed into a multiplex PCR reaction and capillary electrophoresis to uniquely profile our collection of banana cultivars. The developed fingerprinting panel successfully amplified a total of 95 alleles, with 6 to 13 alleles per SSR marker. The average SSR marker polymorphic information content (PIC) value is 0.812, indicating the informativeness of the panel. Analysis of molecular variance (AMOVA) shows that 97% of the molecular variance from our banana collection is due to inter-variety genetic diversity, while the remaining 3% is due to intra-variety genetic diversity. A population structure analysis groups our collection of banana varieties according to the presence of at least one M. balbisiana (B) genome in their genetic makeup. The Cavendish variety, however, showed a distinct structure compared to the other cultivars. This SSR fingerprinting panel provides valuable insights into the genetic diversity and relatedness between banana cultivars in Malaysia. It has the potential to assist future banana breeding initiatives and serve as an effective quality control measure for verifying varieties in a tissue culture facility involved in banana planting materials production.

Evaluation of an E6/E7 PCR-capillary electrophoresis fragment analysis in the genotyping of human papillomavirus in archival FFPE samples of oropharyngeal cancer.

Accumulating evidence has demonstrated that high-risk human papillomaviruses (HR-HPVs) are involved in the etiology of a subset of oropharyngeal squamous cell carcinoma (OPSCC). In this regard, the International Agency for Research on Cancer (IARC) has recommended direct molecular HPV testing. So far, there is no agreement on the most appropriate method for HPV detection on OPSCC formalin-fixed paraffin-embedded (FFPE) materials. In this study, we aimed to evaluate the performance of the high-sensitive SureX HPV assay in OPSCC FFPE tissues compared with LiPA-25 and p16ink4a immunostaining. A retrospective series of FFPE primary OPSCC cases were diagnosed between 2008 and 2019 and provided by the Henan Cancer Hospital, China. The level of agreement of two assays was determined using Cohen's Kappa (κ) statistics. A total of 230 FFPE OPSCC samples from tumor resections (n = 160) and diagnostic biopsies (n = 70) were detected. Sixty-six (28.7%) and 70 (30.4%) samples were identified as HPV-DNA-positive by LiPA-25 and SureX, respectively, of which HPV16 was largely the most common type (95.5% vs 94.3%). We found a perfect concordance between LiPA-25 and SureX for HPV-DNA status (κ = 0.906, 95% CI: 0.875-0.937) and for HPV16 (κ = 0.925, 95% CI: 0.897-0.953). In addition, SureX and p16ink4a immunostaining had a perfect concordance (κ = 0.917, 95% CI: 0.888-0.946). Moreover, the HPV-driven fraction, based on double positivity for HPV-DNA and p16ink4a, was similar between SureX (63 of 230, 27.4%) and LiPA-25 (60 of 230, 26.1%). Similar results were found in samples from resections and biopsies. SureX and LiPA-25 are comparable. SureX could be used for routine HPV-DNA detection and genotyping on archival OPSCC FFPE tissues.

Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

Comparison of multiplex PCR capillary electrophoresis assay and PCR-reverse dot blot assay for human papillomavirus DNA genotyping detection in cervical cancer tissue specimens.

The study aimed to evaluate the positivity rates and genotype distribution of the multiplex PCR capillary electrophoresis (MPCE) and PCR-Reverse Dot Blot (PCR-RDB) assays for human papillomavirus (HPV) detection in cervical cancer tissue specimens, and to explore their detection principles and applications in large-scale population screening. The MPCE and PCR-RDB assays were performed separately on 425 diagnosed cervical cancer tissue specimens. Subsequently, the results of both assays were compared based on the HPV infection positivity rates and genotype distribution. The overall positive rates of HPV genotypes for the MPCE and PCR-RDB assays were 97.9% and 92.9%, respectively. A p-value < 0.001 indicated a statistically significance difference in consistency between the two assays. The kappa value was 0.390, indicating that the consistency between both assays was fair. HPV16 was the most common single-genotype infection type, with infection rates detected via MPCE and PCR-RDB assays being 75.7% and 68.3%, respectively. In the age group >50 years, the HPV multiple-type infection rate detected via MPCE assay was significantly higher than that detected by the PCR-RDB assay, with a statistically significant difference (p = 0.002). To reduce the false-negative rate and improve screening efficiency, the MPCE assay, which targets the oncogenic gene E6/E7 segments, can be extended to the general female population for the early detection, diagnosis, and treatment of cervical cancer.

Identification of Comprehensive Biomarkers in Patients With Mismatch Repair-Deficient Colon Adenocarcinoma Based on Parallel Multiomics

Immunocheckpoint inhibitors have shown impressive efficacy in patients with colon cancer and other types of solid tumor that are mismatch repair-deficient (dMMR). Currently, PCR-capillary electrophoresis is one of the mainstream detection methods for dMMR, but its accuracy is still limited by germline mismatch repair (MMR) mutations, the functional redundancy of the MMR system, and abnormal methylation of MutL Homolog 1 promoter. Therefore, this study aimed to develop new biomarkers for dMMR based on artificial intelligence (AI) and pathologic images, which may help to improve the detection accuracy. To screen for the differential expression genes (DEGs) in dMMR patients and validate their diagnostic and prognostic efficiency, we used the expression profile data from the Cancer Genome Atlas (TCGA). The results showed that the expression of Immunoglobulin Lambda Joining 3 in dMMR patients was significantly downregulated and negatively correlated with the prognosis. Meanwhile, our diagnostic models based on pathologic image features showed good performance with area under the curves (AUCs) of 0.73, 0.86, and 0.81 in the training, test, and external validation sets (Jiangsu Traditional Chinese Medicine Hospital cohort). Based on gene expression and pathologic characteristics, we developed an effective prognosis model for dMMR patients through multiple Cox regression analysis (with AUC values of 0.88, 0.89, and 0.88 at 1-, 3-, and 5-year intervals, respectively). In conclusion, our results showed that Immunoglobulin Lambda Joining 3 and nucleus shape–related parameters (such as nuclear texture, nuclear eccentricity, nuclear size, and nuclear pixel intensity) were independent diagnostic and prognostic factors, suggesting that they could be used as new biomarkers for dMMR patients.

Poly-G for tumor matched samples chronicles the evolution of human colorectal cancer

Objective: To analyze poly-guanine (poly-G) genotypes and construct the phylogenetic tree of colorectal cancer (CRC) and provide an efficient and convenient method for the study of intra-tumor heterogeneity and tumor metastasis pathway. Methods: The clinicopathological information of patients with primary colorectal cancer resection with regional lymph node metastases were retrospectively collected in the Department of General Surgery, General Hospital of Tianjin Medical University from January 2017 to December 2017. The paraffin sections of the paired tumor samples were performed consecutively, and multi-region microdissection was performed after histogene staining. The phenol-chloroform extraction and ethanol precipitation scheme was used to obtain DNA, and Poly-G multiplex PCR amplification and capillary electrophoresis detection were performed. The correlation between Poly-G mutation frequency and clinicopathological parameters was analyzed. Based on the difference of Poly-G genotypes between paired samples, the distance matrix was calculated, and the phylogenetic tree was constructed to clarify the tumor metastasis pathway. Results: A total of 237 paired samples were collected from 20 patients including 134 primary lesions, 66 lymph node metastases, 37 normal tissues, and Poly-G mutation was detected in 20 patients (100%). The mutation frequency of Poly-G in low and undifferentiated patients was (74.10±23.11)%, higher than that in high and medium differentiated patients [(31.36±12.04)%, P<0.001]. In microsatellite instability patients, the mutation frequency of Poly-G was (68.19±24.80)%, which was higher than that in microsatellite stable patients [(32.40±14.90)%, P=0.003]. The Poly-G mutation frequency was not correlated with age, gender, and pathological staging (all P>0.05). Based on Poly-G genotype difference of the paired samples, the phylogenetic trees of 20 patients were constructed, showing the evolution process of the tumor, especially the subclonal origins of lymph node metastasis. Conclusion: Poly-G mutations accumulate in the occurrence and development of CRC, and can be used as genetic markers to generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

Analysis of the clinical features and the risk factors of severe human metapneu movirus-associated community acquired pneumonia in children

Objective: To investigate the clinical characteristics and the risk factors of severe human metapneumovirus (hMPV)-associated community acquired pneumonia (CAP) in children. Methods: A retrospective case summary was conducted. From December 2020 to March 2022, 721 children who were diagnosed with CAP and tested positive for hMPV nucleic acid by PCR-capillary electrophoresis fragment analysis of nasopharyngeal secretions at the Yuying Children's Hospital, the Second Affiliated Hospital of Wenzhou Medical University were selected as the research objects. The clinical characteristics, epidemiological characteristics and mixed pathogens of the two groups were analyzed. According to CAP diagnostic criteria, the children were divided into the severe group and the mild group. Chi-square test or Mann-Whitney rank and contrast analysis was used for comparison between groups, while multivariate Logistic regression was applied to analyze the risk factors of the severe hMPV-associated CAP. Results: A total of 721 children who were diagnosed with hMPV-associated CAP were included in this study, with 397 males and 324 females. There were 154 cases in the severe group. The age of onset was 1.0 (0.9, 3.0) years, <3 years old 104 cases (67.5%), and the length of hospital stay was 7 (6, 9) days. In the severe group, 67 children (43.5%) were complicated with underlying diseases. In the severe group, 154 cases (100.0%) had cough, 148 cases (96.1%) had shortness of breath and pulmonary moist rales, and 132 cases (85.7%) had fever, 23 cases (14.9%) were complicated with respiratory failure. C-reactive protein (CRP) was elevated in 86 children (55.8%), including CRP≥50 mg/L in 33 children (21.4%). Co-infection was detected in 77 cases (50.0%) and 102 strains of pathogen were detected, 25 strains of rhinovirus, 17 strains of Mycoplasma pneumoniae, 15 strains of Streptococcus pneumoniae, 12 strains of Haemophilus influenzae and 10 strains of respiratory syncytial virus were detected. Six cases (3.9%) received heated and humidified high flow nasal cannula oxygen therapy, 15 cases (9.7%) were admitted to intensive care unit, and 2 cases (1.3%) received mechanical ventilation. In the severe group, 108 children were cured, 42 children were improved, 4 chlidren were discharged automatically without recovery and no death occurred. There were 567 cases in the mild group. The age of onset was 2.7 (1.0, 4.0) years, and the length of hospital stay was 4 (4, 6) days.Compared with the mild group, the proportion of children who age of disease onset <6 months, CRP≥50 mg/L, the proportions of preterm birth, congenital heart disease, malnutrition, congenital airway malformation, neuromuscular disease, mixed respiratory syncytial viruses infection were higher (20 cases (13.0%) vs. 31 cases (5.5%), 32 cases (20.8%) vs. 64 cases (11.3%), 23 cases (14.9%) vs. 44 cases (7.8%), 11 cases (7.1%) vs. 18 cases (3.2%), 9 cases (5.8%) vs. 6 cases (1.1%), 11 cases (7.1%) vs. 12 cases (2.1%), 8 cases (5.2%) vs. 4 cases (0.7%), 10 cases (6.5%) vs. 13 cases (2.3%), χ2=0.42, 9.45, 7.40, 4.94, 11.40, 8.35, 3.52, 6.92, all P<0.05). Multivariate Logistic regression analysis showed that age<6 months (OR=2.51, 95%CI 1.29-4.89), CRP≥50 mg/L (OR=2.20, 95%CI 1.36-3.57), prematurity (OR=2.19, 95%CI 1.26-3.81), malnutrition (OR=6.05, 95%CI 1.89-19.39) were the independent risk factors for severe hMPV-associated CAP. Conclusions: Severe hMPV-associated CAP is most likely to occur in infants under 3 years old and has a higher proportion of underlying diseases and co-infection. The main clinical manifestations are cough, shortness of breath and pulmonary moist rales, fever. The overall prognosis is good. Age<6 months, CRP≥50 mg/L, preterm birth, malnutrition are the independent risk factors for severe hMPV-associated CAP.

Application of multiplex PCR capillary electrophoresis and analysis of distribution characteristics of HPV infection subtypes

Objective: To analyze the characteristics of human papillomavirus (HPV) infection and the distribution of HPV subtypes in Shijiazhuang, Hebei Province, and to explore the application evaluation of multiple PCR capillary electrophoresis fragment analysis for HPV typing test. Methods: A population-based cross-sectional study was conducted among 434 women (age range 17 to 74 years old, 260 patients and 174 physical examinations) included from May to August 2022 in Hebei General Hospital. HPV typing was detected by multiple PCR-capillary electrophoresis fragment analysis. Using the multiple fluorescence quantitative PCR kit as a reference, Chi-square test was used to analyze the diagnostic effect of multiple PCR-capillary electrophoresis fragment analysis, and the consistency was analyzed by Kappa value. Results: The total HPV infection rate was 45.85%(199/434), including 35.48% (154/434) of high-risk HPV (HR-HPV), 3.92% (17/434) of low-risk HPV (LR-HPV), 6.45% (28/434) of HR-HPV and LR-HPV mixed infection, 27.88% (121/434) of single type HPV and 17.97% (78/434) of multi type HPV. HPV52 (9.68%, 42/434), HPV16 (6.91%, 30/434), and HPV58 (6.91%, 30/434) are common HPV subtypes. The positive rate of physical examination was 45.40% (79/174), which was slightly lower than that of patients 46.15% (120/260), there was no significant difference (χ2=0.024,P>0.05). The highest infection rate in the 17-30 age group was 54.76% (46/84), and there was no statistical difference among the age groups(χ2=4.123,P>0.05). The sensitivity and specificity of multiplex PCR capillary electrophoresis fragment analysis were 92.96% and 94.04%, respectively, and Kappa value was 0.870, with the multiplex fluorescent quantitative PCR as the reference. Conclusion: HPV infection may appear younger, and the positive rate of HR-HPV infection is the highest, with HPV52, 16, 58 as the main infection subtypes. The detection results of multiplex PCR capillary electrophoresis fragment analysis method are highly consistent with those of multiplex fluorescent quantitative PCR method, which is suitable for HPV DNA typing.

The incidence and clinical characteristics of fragile X syndrome in China.

Fragile X syndrome (FXS) is a X-linked neurodevelopmental disorder (NDD). This study aims to investigate the incidence of FXS in Chinese children and analyze the comprehensive clinical characteristics of these FXS children. Children diagnosed with idiopathic NDD were recruited between 2016 and 2021 from the department of Child Health Care, Children's Hospital of Fudan University. We combined tetraplet-primed PCR-capillary electrophoresis and whole exome sequencing (WES)/panel or array-based comparative genomic hybridization (array-CGH) to identify the size of the CGG repeats and the mutations or copy number variations (CNVs) in the genome and in FMR1. The clinical features of FXS children were analyzed according to pediatricians' recording, parental questionnaires, the results of examinations and follow-up. The incidence of FXS in Chinese children with idiopathic NDD was 2.4% (42/1753) and in those with FXS, 2.38% had a deletion (1/42). Here, we present the clinical characteristics of 36 children with FXS. Overweight was observed in two boys. The average intelligence quotient (IQ)/development quotient (DQ) of all FXS patients was 48. The average ages of meaningful words and walking alone were 2 years and 10 months and 1 year and 7 months, respectively. The most frequent repetitive behavior was stimulated by hyperarousal to sensory stimulation. On social aspects, social withdrawal, social anxiety, and shyness accounted for 75%, 58%, and 56% of the total number of children, respectively. Approximately 60% of FXS children in this cohort were emotionally labile and prone to temper tantrums. Self-injury and aggression toward others could also be observed, at 19% and 28%, respectively. The most frequent behavioral problem was attention-deficit hyperactivity disorder (ADHD) seen in 64% and the most common facial features were a narrow and elongated face and large or prominent ears in 92% of patients. Screening of FMR1 full mutation provides the possibility for patients' further medical supports and the clinical features of FXS children obtained in this study will increase the understanding and diagnosis of FXS.

An innovative single-base extension method for synchronous detection of point mutations and MSI status in colorectal cancer.

An accurate genotyping analysis is one of the critical prerequisites for patients with colorectal cancer receiving matched therapies. Conventional genotyping analysis is currently used to detect either gene mutations or MSI status, delaying the detection of critical tumor biomarkers and thus the optimal time for treatment. An assay that analyzes both biomarkers in a streamlined process is eagerly needed. We developed an assay combining Multiplex PCR Amplification, Single-base Extension and capillary electrophoresis (CE) analysis (MASE-CE) for synchronous detection of KRAS/NRAS/BRAF mutations and MSI status. In a 190 colorectal cancer cohort, we identified seven somatic mutations in KRAS, NRAS and BRAF as well as five MSI loci (D2S123/D5S346/D17S250/BAT-25/BAT-26) simultaneously. KRAS/NRAS/BRAF mutations were detected by NGS and MASE-CE, and MSI status were detected by PCR-CE and MASE-CE methods. The MASE-CE method showed high consistency with NGS for mutation detection (Kappa value ≥0.8) and PCR-CE (Kappa value=0.79). In addition, the limits of detection (LOD) of MASE-CE assay for MSI and somatic mutation were 5% and 2%, respectively. In somatic mutation detection and MSI detection, the LOD of MASE-CE assay was superior to that of qPCR and NGS. MASE-CE assay is a highly sensitive, time-saving and specimen-saving method, which can greatly avoid the cumbersome testing process and provide clinical decision for doctors in time.

An Accurate and Rapid Way for Identifying Food Geographical Origin and Authenticity: Editable DNA-Traceable Barcode.

DNA offers significant advantages in information density, durability, and replication efficiency compared with information labeling solutions using electronic, magnetic, or optical devices. Synthetic DNA containing specific information via gene editing techniques is a promising identifying approach. We developed a new traceability approach to convert traditional digitized information into DNA sequence information. We used encapsulation to make it stable for storage and to enable reading and detection by DNA sequencing and PCR-capillary electrophoresis (PCR-CE). The synthesized fragment consisted of a short fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene from the Holothuria fuscogilva (ID: LC593268.1), inserted geographical origin information (18 bp), and authenticity information from Citrus sinensis (20 bp). The obtained DNA-traceable barcodes were cloned into vector PMD19-T. Sanger sequencing of the DNA-traceable barcode vector was 100% accurate and provided a complete readout of the traceability information. Using selected recognition primers CAI-B, DNA-traceable barcodes were identified rapidly by PCR amplification. We encapsulated the DNA-traceable barcodes into amorphous silica spheres and improved the encapsulation procedure to ensure the durability of the DNA-traceable barcodes. To demonstrate the applicability of DNA-traceable barcodes as product labels, we selected Citrus sinensis as an example. We found that the recovered and purified DNA-traceable barcode can be analyzed by standard techniques (PCR-CE for DNA-traceable barcode identification and DNA sequencing for readout). This study provides an accurate and rapid approach to identifying and certifying products' authenticity and traceability.

Identifying Human Remains from 20th Century Warfare: A State of the Field Essay.

As we continually reflect on the wars of the 20th century, identification of the remains of victims takes an increasingly prominent position in ongoing research. Existing work on the identification of human remains from 20th century wars primarily covers the determination of phenotypic characteristics, kinship and geographic origins, supporting the establishment of genetic information databases. Compared with standard forensic methods, DNA analyses have revealed greater effectiveness. The process of DNA analysis includes DNA extraction, genetic marker testing and data analysis. Protocols from ancient DNA research can be applied to degraded remains, and next-generation sequencing (NGS) techniques can compensate for shortcomings in the most commonly-used PCR-capillary electrophoresis typing. As it stands, wide-ranging inter-governmental and inter-institutional collaboration is necessary in order to set up NGS-based public databases, and thereby promote the identification of human remains and archaeological forensics.

Genotyping of Plasmodium falciparum to Assess Clone Composition in Parasite Cultures.

Defining clone composition in Plasmodium falciparum cultures is key to verify that in vitro experiments are performed on the parasite line of interest. Genotyping of the highly polymorphic merozoite surface protein 2 gene(msp2) is a widely established method to define P. falciparum clones. Specific size variants from the two msp2 families (IC and FC27) can be used as "fingerprints" to identify individual clones in parasite mixtures. Size variant genotyping of msp2 using fluorescent nested PCR followed by fragment analysis by capillary electrophoresis (CE) provides accurate information about the presence of one or multiple parasite clones. Here, we describe an adaptation of this approach to assess the integrity and purity of P. falciparum lines kept in in vitro culture. In addition, we describe the use of synthetic mock parasite mixtures with the msp2 sequences from the parasite lines kept in culture that can provide a good estimate of the assay sensitivity, specificity, and reproducibility. We suggest that genotyping of P. falciparum lines should be performed ona regular basis as part ofthe standard procedures of in vitro parasite culture, as a way to secure that the parasite lines of interest are cultivated, and to monitor any cross-contamination and/or recombination events.

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis.

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

A Ge.F.I. – ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists – International Society of Forensic Genetics (Ge.F.I. – ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of −1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

A new minisatellite VNTR marker, Pscp1, discovered for the identification of opium poppy

Opium poppy, a member of the Papaveraceae family, is an ancient herbaceous plant and well-known medical resource in the pharmaceutical industry. However, opium poppies are grown worldwide for producing illicit drugs, significantly increasing the incidence of narcotic drug abuse. Since the narcotic poppy has not yet been genetically investigated, we characterized a novel variable number tandem repeat (VNTR) marker of forensically important poppy species based on the genetic analysis of 164 samples collected from two locations spanning the Jeolla province and Jeju island of South Korea. Comparing analysis of the chloroplast (cp) genome sequences for four representative species of Papaver (Papaver somniferum, Papaver somniferum subs. setigerum, Papaver orientale, and Papaver rhoeas) revealed a unique region with 1–3 repeats for 16 nucleotide motifs in the genome inverted repeat A (IRA, positions 128,651 to 128,698) region. For 16 nucleotide motifs, 3 repeats were found in P. somniferum, and 2 repeats were found in P. somniferum subs. setigerum. Therefore, 10 known and the 133 unknown, seized Papaver species were compared to determine whether the species could be identified via variations in the repeat units. The sizes of a novel VNTR ranged from 181 to 252 bp between the species. Phylogenetic analysis confirmed that a novel VNTR, which we named Pscp1, could clearly distinguish between the narcotic and non-narcotic types of Papaver species based on the patterns of sequence variation. Interestingly, we found that Pscp1 could also distinguish between P. somniferum and P. somniferum subs. setigerum. The regions of eight non-narcotic species displayed similar patterns and also differences were found due to the nucleotide substitution and deletion events. The structural differences of Pscp1 were observed within the two narcotic species or between the narcotic and non-narcotic species, suggesting that these variations may act as a genetic marker. We, therefore, developed a new Pscp1 PCR-capillary electrophoresis (CE) method that can reliably identify the narcotic type of Papaver species. Taken together, our findings suggest that the newly developed Pscp1 can be used as an identification marker of opium poppy, and establish that the Pscp1 genotyping method by PCR-CE is an effective primary screening tool that can also contribute to species discrimination in the field of forensic diagnosis and applications.

Complementarity between microhistological analysis and PCR-capillary electrophoresis in diet analysis of goats and cattle using faecal samples

An evaluation is made of the complementarity between two non-invasive techniques, cuticle microhistological analysis (CMA) and PCR-capillary electrophoresis (PCR-CE) DNA-based analysis, for the determination of herbivore diet composition from faecal samples. Cuticle microhistological analysis is based on the different microanatomical characteristics of the epidermal fragments remaining in the faeces. The PCR-CE technique combines PCR amplification of a trnL(UAA) genomic DNA region with amplicon length determination by CE, with this length being characteristic for each species or taxon. A total of 37 fresh stool samples were analyzed, including 16 from feral goats (Capra hircus) from the Tramuntana mountain range (Mallorca, Baleares) and 11 from Bruna dels Pirineus cattle breed (Bos taurus) from the surrounding Montserrat mountain range (Barcelona, Spain). All the animals were in a free grazing Mediterranean pine habitat, dominated by Aleppo pine (Pinus halepensis). The results showed that both techniques detected a similar number of plant components in the faeces of goats and cows. In the case of goats, a positive correlation was obtained between the percentage of samples in which a particular taxon is detected by CMA and the percentage of samples in which that taxon is detected by PCR-CE. This correlation was not observed in the case of cows. It is concluded that PCR-CE is a fast and reliable method to detect the different plant components in the faeces of herbivores. However, it cannot be considered as an alternative to CMA, but as a complementary method, since both techniques can detect some taxa that are not detected by the other technique. In addition, CMA detected the presence of the different taxa in a greater number of samples, and at the same time, it enables quantitative data to be obtained for plant diet composition. The species of herbivore also seems to influence the results obtained by PCR-CE, so more studies are required to address this aspect.

Detection of JAK2V617F and CALR Gene Mutations by Multiplex of Patients with Myeloproliferative Neoplasms PCR-Capillary Electrophoresis

To evaluate the proformance of multiplex PCR and capillary electrophoresis(MPCE) in the detection of JAK2V617F and CALR mutation in myeloproliferative neoplasms(MPN). The specificity primers of JAK2617F gene mutation and the primers of CALR gene were designed at the same time. The JAK2V617F and CALR gene primers were labeled with Cy5 fluorescence, all the primers were mixed in one tube for multiplex PCR and the PCR prodcuts were analysised by capillary electrophoresis. Then detection limit and sensitivity of MPCE were evaluated, and compared with comercial diagnostic kit. JAK2V617F and CALR gene mutations could be detect by MPCE in one PCR test. JAK2V617F mutation could be detected at 0.01 ng genomic DNA, double positive JAK2V617F and CLAR gene mutations could be detected at 0.1 ng genomic DNA, at least 0.1% JAK2V617F positive mutation could be detected. The consistency between MPCE and commercial diagnostic gene mutation kit was 100%. It is developed that a new gene mutation detection method of JAK2 V617F and CLAR gene based on MPCE in our experiment and it can be used as a new reagent for molecular diagnosis of MPN patients.