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.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) have emerged as critical agents for cancer therapy. By inhibiting the catalytic activity of PARP enzymes and trapping them in the DNA, PARPis disrupt DNA repair, ultimately leading to cell death, particularly in cancer cells with homologous recombination repair deficiencies, such as those harboring BRCA mutations. This review delves into the mechanisms of action of PARPis in anticancer treatments, including the inhibition of DNA repair, synthetic lethality, and replication stress. Furthermore, the clinical applications of PARPis in various cancers and their adverse effects as well as their combinations with other therapies and the mechanisms underlying resistance are summarized. This review provides comprehensive insights into the role and mechanisms of PARP and PARPis in DNA repair, with a particular focus on the potential of PARPi-based therapies in precision medicine for cancer treatment.
Humans ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Neoplasms/genetics* ; DNA Repair/drug effects* ; Animals ; Antineoplastic Agents/therapeutic use*

OBJECTIVE: To confirm the sequence of a null allele HLA-C*08:127N produced by a base insertion. METHODS: PCR sequence-specific oligonucleotide probe (SSOP) and PCR sequence-based typing (SBT) were used for HLA routine detection, which discovered abnormal sequence maps of HLA-C in one acute myeloid leukemia patient. The sequence of the above loci was confirmed by next generation sequencing (NGS) technology. RESULTS: The SSOP typing result showed that HLA-C locus was C*03:04, C*08:01, while the sequence was suspected to be inserted or deleted in exon 3 by SBT, and finally confirmed by NGS as C*03:04, C*08:127N. CONCLUSION When base insertion produces HLA null alleles, SBT analysis software cannot provide correct results, but NGS technology can more intuitively obtain accurate HLA typing results.
Humans ; Alleles ; High-Throughput Nucleotide Sequencing ; HLA-C Antigens/genetics* ; Histocompatibility Testing ; Polymerase Chain Reaction ; Leukemia, Myeloid, Acute/genetics* ; Sequence Analysis, DNA ; Mutagenesis, Insertional ; Exons

OBJECTIVES: To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. METHODS: L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. RESULTS: The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. CONCLUSIONS The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.
Listeria monocytogenes/isolation & purification* ; Gold Colloid ; Bacterial Toxins/genetics* ; Polymerase Chain Reaction/methods* ; Hemolysin Proteins/genetics* ; Bacterial Proteins/genetics* ; DNA, Bacterial/genetics* ; Food Microbiology ; Heat-Shock Proteins

Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease Ⅰ for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease Ⅰ-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.
Genome, Viral/genetics* ; Polymerase Chain Reaction/methods* ; DNA, Viral/genetics* ; Bacteriophage T7/enzymology* ; Synthetic Biology/methods*

In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3'→5' exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.
DNA-Directed DNA Polymerase/metabolism* ; Bacillus Phages/genetics* ; Metagenome

Calcined oyster is a commonly used shellfish traditional Chinese medicine in clinical practice in China. During the processing of oysters, their microscopic characteristics are destroyed, and open-fire calcination can damage the DNA of oysters, making it difficult to identify the primary source. The establishment of a specific polymerase chain reaction(PCR) method for the identification of calcined oysters can provide a guarantee for the safety and clinical efficacy of the medicine and its processed products. With Ostrea gigas as an example, the DNA extraction method of decoction pieces and formula particles of calcined oysters was improved, and high-quality DNA was obtained. Based on the specific single nucleotide polymorphism(SNP) sites of O. gigas and the other two species, the specific identification primers were designed, and the site-specific identification method of formula granules of calcined oyster(O. gigas) was established. The specificity and applicability of the method were investigated. The results showed that when the annealing temperature was 54 ℃, and the cycle was 44 times, the PCR amplified products of calcined oyster(O. gigas) and its formula granules produced a single bright identification band at 102 bp, while the other two species of oysters, O. talienwhanensis Crosse and O. rivularis Gould, had no band. In this study, DNA extraction and PCR identification of animal medicinal materials by calcination were established for the first time, which provided a tool for solving the difficult identification of calcined decoction pieces and ensuring drug safety.
Animals ; Ostrea/classification* ; Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; DNA/genetics*

To promote the conservation and utilization of the germplasm resources and provide a basis for the breeding of new varieties of Murraya paniculata, this study analyzed the genetic diversity of the germplasm resources and developed the molecular identity(ID) card of M. paniculata. Multiple fluorescence PCR-capillary electrophoresis was performed for 65 germplasm accessions of M. paniculata based on 9 SSR markers identified from the M. paniculata genome, and the molecular weights and alleles of the amplified bands were analyzed. According to the banding patterns of the 9 SSR primers, this study analyzed the genetic diversity of each germplasm accession of M. paniculata and developed molecular ID cards for the test samples. The results showed that 9 pairs of SSR primers detected 78 alleles, with an average of 8.67 alleles. The observed and expected heterozygosity was 0.338-0.831(average of 0.601) and 0.413-0.853(average of 0.721), respectively. The Shannon's information index varied within the range of 0.880-1.994, with an average of 1.41. The polymorphic information content was within the range of 0.391-0.835, with an average of 0.696, which indicated rich genetic diversity. When the genetic identity was 0.347, the 65 germplasm accessions were classified into 5 groups. Based on the results, this study employed the 5 SSR primers with higher polymorphisms to develop the molecular ID cards for the germplasm resources of M. paniculata and created QR code ID cards for the 49 core germplasm accessions preserved in the Yunfu germplasm nursery, laying a foundation for the new variety breeding, production, utilization, and traceability of M. paniculata.
Microsatellite Repeats ; DNA, Plant/genetics* ; Murraya/classification* ; Genetic Variation ; Alleles ; Polymerase Chain Reaction ; Polymorphism, Genetic

OBJECTIVE: To analyze the serological characteristics and molecular mechanism for an individual with p phenotype. METHODS: An individual with p phenotype upon blood group identification at Jiaxing Blood Center in May 2021 was analyzed. ABO, RhD and P1PK blood groups and irregular antibodies in her serum were identified using conventional serological methods. The encoding region of α1, 4-galactosyltransferase gene (A4GALT) encoding P1 and Pk antigens was analyzed by polymerase chain reaction-sequence-based typing (PCR-SBT). RESULTS: The individual was A group, RhD positive and had a p phenotype of the P1PK blood group system. Anti-PP1Pk was discovered in her serum. Sequencing analysis revealed that she has harbored a homozygous c.343A>T variant of the A4GALT gene. CONCLUSION The homozygous c.343A>T variant of the A4GALT gene probably underlay the p phenotype in this individual.
Female ; Animals ; Blood Group Antigens ; Homozygote ; Phenotype ; Polymerase Chain Reaction ; Sequence Analysis, DNA

Objective To investigate the effect of blood group serology and polymerase chain reaction with sequence-specific primers (PCR-SSP) on identification and genotyping of ambiguous ABO blood group. Methods Eighty suspicious ABO blood group samples were identified by serology and polymerase chain reaction with sequence-specific primers (PCR-SSP). The final blood group type and the strategy of the transfusion of each case were determined according to the results of serology and PCR-SSP. Results 40 cases were confirmed to be subtypes, and the remaining 40 cases were normal types with weakened antigens or missing antibodies due to other reasons. The results of molecular genetic blood group typing based on PCR-SSP were 41 cases of subtypes (There were 3 discrepancies between two methods: one was Ael identified by serological methods, while its gene type was O2O2; one was common type O, while its gene type was BO1; one was type A, while its gene type was AB.) and 39 cases of normal ones. Conclusion Genotyping technology combined with serological typing has an important significance in identification of ABO blood groups.
ABO Blood-Group System/genetics* ; Genotype ; Polymerase Chain Reaction ; Antibodies ; DNA Primers