BACKGROUND AND OBJECTIVE

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19 has significantly challenged the public health landscape in late 2019. After almost 3 years of the first ever SARS-CoV-2 case, the World Health Organization (WHO) declared the end of this global health emergency in May 2023. Although, despite the subsequent drop of COVID-19 cases, the SARS-CoV-2 infection still exhibited multiple waves of infection, primarily attributed to the appearance of new variants. Five of these variants have been classified as Variants of Concern (VOC): Alpha, Beta, Gamma, Delta, and the most recent, Omicron. Therefore, the development of methods for the timely and accurate detection of viral variants remains fundamental, ensuring an ongoing and effective response to the disease. This study aims to evaluate the feasibility of the application of an in-house approach in genomic surveillance for the detection of SARS-CoV-2 variants using in silico designed primers.

The primers used for the study were particularly designed based on conserved regions of certain genes in the virus, targeting distinct mutations found in known variants of SARS-CoV-2. Viral RNA extracts from nasopharyngeal samples (n=14) were subjected to quantitative and qualitative tests (Nanodrop and AGE). Selected samples were then analyzed by RT-PCR and amplicons were submitted for sequencing. Sequence alignment analysis was carried out to identify the prevailing COVID-19 variant present in the sample population.

The study findings demonstrated that the in-house method was able to successfully amplify conserved sequences (spike, envelope, membrane, ORF1ab) and enabled identification of the circulating SARS-CoV-2 variant among the samples. Majority of the samples were identified as Omicron variant. Three out of four designed primers effectively bound into the conserved sequence of target genes present in the sample, revealing the specific SARSCoV-2 variant. The detected mutations characterized for Omicron found in the identified lineages included K417N, S477N, and P681H which were also identified as mutations of interest. Furthermore, identification of the B.1.448 lineage which was not classified in any known variant also provided the potential of the developed in-house method in detecting unknown variants of COVID-19.

Among the five VOCs, Omicron is the most prevalent and dominant variant. The in-house direct PCR product sequencing surveillance (DPPSS) method provided an alternative platform for SAR-CoV-2 variant analysis which is accessible and affordable than the conventional diagnostic surveillance methods and the whole genome sequencing. Further evaluation and improvements on the oligonucleotide primers may offer significant contribution to the development of a specific and direct PCRbased detection of new emerging COVID-19 variants.

The amplification of c-myc oncogene was evaluated in 42 cases of ovarian carcinomas to correlate with clinical parameters. Using oligonucleotide primers, sequences from the c-myc exon-3 gene and from a control gene, tissue plasminogen activator (tPA), were amplified simultaneously by polymerase chain reaction (PCR). After the products of differential PCR (d-PCR) were electrophoresed, slot blot hybridization was performed, and hybridized with P32 dATP-labeled myc and tPA oligonucleotide probes and then autoradiographed. The signal intensities of the two products were quantitated by densitometry and the ratios of two products (c-myc/tPA) were measured. The ovarian carcinomas showed significantly increased amplification of c-myc oncogene Oligonucleoti compared to normal control group (p<0.05). 15 of 42 cases (35.7%) showed various degrees of the MYC gene amplification up to 27 folds in various histologic types of ovarian carcinomas. No significant differences of the MYC gene amplification according to histologic subtypes, tumor action) grades and clinical stages of ovarian carcinomas were present.
Densitometry ; DNA Primers ; Genes, myc ; Oligonucleotide Probes ; Oncogenes* ; Polymerase Chain Reaction* ; Tissue Plasminogen Activator

Genetic analysis from forensic microsamples is a urgent, difficult task in forensic science, because it is frequently limited by the amount of specimen available in forensic practice, much effort has been carried out to resolve this difficulty. Whole genome amplification (WGA) technology, which was developing quickly in these years, has been thought to be a powerful, reliable and efficient strategy in analysis of minute amount DNA on many fields. In this review, we discuss its application in forensic science.
DNA/isolation & purification* ; DNA Primers ; Forensic Medicine/methods* ; Genome, Human ; Humans ; Nucleic Acid Amplification Techniques ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction/methods* ; Sequence Analysis, DNA

OBJECTIVETo develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray.

METHODSTwo sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1).

RESULTSA combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity.

CONCLUSIONA combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

OBJECTIVE: ABO genotyping for forensic identification by oligonucleotide chip. METHODS: Oligonucleotide microarrays which could detect 3 different SNPs in exon 6 and exon 7 for ABO genotyping were used. Population studies on ABO was carried out in a sample of 115 unrelated Chinese Han individuals. The method was also applied to cases. RESULTS: The technique could identify 6 genotypes of ABO system. According to the results of population studies, no significant deviations from Hardy-Weinberg equilibrium could be found. The observed and expected heterozygosities were 0.591 and 0.616 respectively. The polymorphic information content was 0.544. The average exclusion probabilities in buos and trios was 0.188 and 0.344 respectively. The discrimination power is 0.777. CONCLUSION The data and case application demonstrated that ABO typing by oligonucleotide probe arrays was a useful technique for paternity testing and individual identification.
ABO Blood-Group System/genetics* ; Blood Stains ; DNA/blood* ; DNA Primers ; Female ; Forensic Medicine ; Genotype ; Hair/chemistry* ; Humans ; Oligonucleotide Array Sequence Analysis

OBJECTIVE: Research on the application feasibility of SNP genotyping for forensic identification by microarrays. METHODS: Oligonucleotide microarrays which could detect 31 different SNPs were used. Population studies on 31 SNP loci was carried out in a sample of 109 unrelated Chinese Han individuals using oligonucleotide microarrays for genotype detection. The method was also applied to cases. RESULTS: No deviations from Hardy-Weinberg equilibrium could be found at the 31 SNP loci 4 loci were medium informative and 27 were high informative. The combination discrimination power (CDP) of the 31 optimal informative SNPs was 0.9999999999979. The matching probability was 2.13 x 10(-12). The average exclusion probability in duos and trios were 0.9609 and 0.9970 respectively. CONCLUSION The data and case application demonstrated that SNP typing by oligonucleotide probe microarrays was a useful technique for paternity testing and individual identification.
Asian People/genetics* ; DNA Fingerprinting/methods* ; DNA Primers ; Forensic Medicine/methods* ; Genotype ; Humans ; Oligonucleotide Array Sequence Analysis ; Paternity ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Tandem Repeat Sequences

The aim of this study was to investigate the factors which affect HLA typing in 311 umbilical cord blood (UCB) samples. The HLA low resolution typing of UCB samples with misinterpreted HLA types from 311 UCB samples analyzed by PCR-SSO and PCR-SSP was performed. 7 samples difficult to determine their HLA genotype were sequenced directly and the reason leading to misinterpret HLA typing was analyzed. The results indicated that 99.4% of misinterpreted samples resulted from the restriction of HLA typing method itself and 0.6% of misinterpreted samples were suspected to be contaminated with maternal blood in UCB. It is concluded that HLA typing is mainly affected by the shortcomings of oligonucleotide probe design for PCR-SSO and lack of allele specific primers of PCR-SSP.
Alleles ; Base Sequence ; DNA Primers ; Fetal Blood ; immunology ; Genotype ; HLA Antigens ; genetics ; Histocompatibility Testing ; methods ; Humans ; Oligonucleotide Probes ; Polymerase Chain Reaction ; methods

In this study, a novel resequencing pathogen microarray (RPM)-based multi-pathogen detection assay was developed to simultaneously detect 14 rotaviruses, 7 caliciviruses, 8 astroviruses, 28 enteroviruses, and 16 rare diarrhea viruses in patients with diarrhea syndrome. The specificity of the assay was examined using confirmed virus-positive specimens, and the sensitivity was evaluated by serial ten-fold dilutions of in vitro transcribed RNA. RPM assay could detect and differentiate virus types/subtypes at 20-2000 copies/microL. The detection threshold of RPM was determined by adjusting the reference concentration, and the detection steps were optimized to type Enterovirus. The nucleic acids of 10 stool samples from patients with unexplained diarrhea were screened, and 6 of them showed positive results. The RPM results were further verified by singleplex PCR followed by sequencing, and no difference was found between the two assays. In conclusion, we have established a high-throughput RPM assay with high specificity and sensitivity, which demonstrates a great potential for the identification of pathogens in patients with unexplained diarrhea and the management of emerging epidemic.
DNA Primers ; genetics ; Diarrhea ; virology ; Feces ; virology ; High-Throughput Screening Assays ; methods ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Sensitivity and Specificity ; Viruses ; classification ; genetics ; isolation & purification

Limitations of polyacrylamide gel or agarose gel electrophoretic methods in genotyping research affect the interpreting of detection results. In order to develop a simple and reliable method for appraising results of ABO genotyping detection, the microfluidic chip analysis system was established by using microfluidic chip to replace the gel electrophoresis and combining with multiplex-PCR-RFLP technique. 150 blood samples were tested by this microfluidic chip analysis system with multiplex-PCR-RFLP technique to evaluate its stability and accuracy. The results showed that all the testing results were consistent with serologic ABO genotyping results and 1 blood sample with decrease of B antigen caused by CML was identified. In conclusion, the established microfluidic chip analysis system is stable and reliable technique. Application of this technique enables the ABO genotyping results to be more objective and accurate.
ABO Blood-Group System ; genetics ; Blood Grouping and Crossmatching ; methods ; DNA Primers ; genetics ; Genotype ; Humans ; Microfluidic Analytical Techniques ; Microfluidics ; Oligonucleotide Array Sequence Analysis

Over a long period of time, studies on HLA structure and function have been the research hotspots. for it is very important to understand the essential of life science and disease mechanism. With the rapid development of molecular biology, HLA typing makes great progress. It has changed from traditional serological typing to DNA-based typing. More and more HLA genotyping methods have been developed and applied. In this essay, the author reviewed and appraised all kinds of HLA genotyping techniques and introduced two new techniques.
DNA Primers ; Genetic Techniques ; Genotype ; HLA Antigens/genetics* ; Histocompatibility Testing/methods* ; Humans ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; Polymerase Chain Reaction/methods* ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA/methods*