Polymerase Chain Reaction Method For Detection Research Articles

Development of a TaqMan polymerase chain reaction detection method for the precise identification and quantification of an attenuated Eimeria maxima vaccine strain in poultry.

Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by Eimeria species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the Eimeria maxima attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve's slope of -3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other E. maxima wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the E. maxima EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method's application. Testing on 61 clinical samples revealed a 57.38% positivity rate for E. maxima species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method's robust stability for the attenuated E. maxima vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

The Use of the Internal Transcribed Spacer Region for Phylogenetic Analysis of the Microsporidian Parasite Enterocytozoon hepatopenaei Infecting Whiteleg Shrimp (Penaeus vannamei) and for the Development of a Nested PCR as Its Diagnostic Tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

The use of the internal transcribed spacer region for phylogenetic analysis of the microsporidian parasite Enterocytozoon hepatopenaei infecting whiteleg shrimp (Penaeus vannamei) and for the development of a nested PCR as its diagnostic tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2%) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Establishment of a multiplex-PCR detection method and development of a detection kit for five animal-derived components in edible meat

A multiplex polymerase chain reaction (PCR) detection method for the simultaneous detection of animal-derived components from deer, cow, sheep, pig and horse in edible meat was established, and a multiplex PCR detection kit for the rapid detection of animal-derived components was developed. According to the mitochondrial cytochrome b (Cyt b) gene of bovine species, sheep species, pig species and horse species and the mitochondrial cytochrome c oxidase subunit I (COX 1) gene of sika deer and red deer as the target gene sequences of primers, the specific primers of five different species were designed, the PCR system was optimized, and the multiplex PCR identification method of five animal-derived components was established. The minimum detection amount was determined by sensitivity test. The results showed that five meat specific amplification bands could be found at the same time in the same reaction system, including 173 bp fragment for venison, 148 bp for beef, 261 bp for pork, 100 bp for mutton and 424 bp for horse, indicating that the method is specific and stable. The minimum detection limit by this method was 1 ng/μL, showing a high sensitivity. According to the different sites in different areas of animal mitochondrial genes, a multiplex PCR detection method was established and a detection kit was developed, and the rapid, sensitive, stable and high-throughput detection of five animal-derived components and adulterated animal components in edible meat can be realized by using the kit.

Analysis of relationship between distribution of pathogenic microorganisms in olfactory cleft and olfactory disorders among patients with upper respiratory inflammation during the prevention and control of COVID-19

Objective: To explore the relationship between pathogens in the olfactory cleft area and olfactory disorders in patients with upper respiratory inflammation (URI) during the prevention and control of 2019 novel coronavirus disease (COVID-19). Methods: A total of 234 URI patients including acute upper respiratory infection, chronic rhinosinusitis (CRS), allergic rhinitis (AR) were continuously selected from September 2020 to March 2021 in Beijing Anzhen Hospital and 98 healthy adults were enrolled as controls. The secretions from the olfactory cleft of all subjects were collected with nasal swabs under nasal endoscopy. Multiple real-time fluorescent quantitative polymerase chain reaction detection method was used to detect nucleic acids of 33 types of respiratory pathogenic microorganism. Sniffin' Sticks olfactory test was performed on all patients with URI. URI patients with olfactory dysfunction were followed up for 9 (8, 10) months (M (Q1, Q3)). SPSS 20.0 software was used for statistical analysis. Results: Among the 98 controls, 9 (9.18%) were positive for pathogenic microorganisms, including 1 (1.02%) rhinovirus, 1 (1.02%) parainfluenza virus type 3, 3 (3.06%) enterovirus, 1 (1.02%) staphylococcus aureus and 3 (3.06%) Moraxella catarrhalis. Among the 234 URI patients, 111 (47.44%) had olfactory disorders and 123 (52.56%) had normal sense of smell. In the olfactory disorder group (111 cases), 38 cases (34.23%) were positive for pathogenic microorganisms, and 4 cases (3.60%) were mixed infection, including 11 cases of rhinovirus (9.91%), 5 cases of coronavirus 229E (4.50%), 2 cases of coronavirus OC43/NL63 (1.80%), 3 cases of parainfluenza virus type 1 (2.70%), 2 cases of enterovirus (1.80%), 1 case of influenza B virus type BV (0.90%), 11 cases of Staphylococcus aureus (9.91%), 7 cases of Moraxella catarrhalis (6.31%), and 1 case of Klebsiella pneumoniae (0.90%). In the normal smell group (123 cases), 18 cases (14.63%) were positive for pathogenic microorganisms, and 1 case (0.81%) was mixed infection, including 3 cases of rhinovirus (2.44%), 4 cases of coronavirus 229E (3.25%), 1 case of Influenza virus type 3 (0.81%), 3 cases of enterovirus (2.44%), 3 cases of Staphylococcus aureus (2.44%), 4 cases of Moraxella catarrhalis (3.25%), and 1 case of Klebsiella pneumoniae (0.81%). Univariate analysis between the two groups found that there were significant differences in the detection rate of pathogenic microorganisms, rhinovirus and Staphylococcus aureus between the groups (all P<0.05). The detection rate of parainfluenza virus type 1, Staphylococcus aureus, and rhinovirus were different between the patients with olfactory disorder and normal olfactory function in the three subgroups of acute upper respiratory tract infection, CRS and AR, respectively (χ2 value was 3.88, 4.53 and 4.73, respectively, all P<0.05). During the follow-up period, among the 111 patients with olfactory disorder, 71 (63.96%) patients' olfactory function returned to normal, 32 (28.83%) patients' olfactory function improved but not completely returned to normal, 8 (7.21%) patients' olfactory function did not improve. Conclusions: During the prevention and control of COVID-19, rhinovirus or Staphylococcus aureus infection or colonization of URI patients is closely related to olfactory disorders. Parainfluenza virus type 1 infection can cause relatively persistent olfactory disorders in patients with acute upper respiratory tract infection. Staphylococcus aureus and rhinovirus colonization are related to the occurrence of olfactory dysfunction in CRS and AR patients respectively.

Correlation of circulating tumor DNA EGFR mutation levels with clinical outcomes in patients with advanced lung adenocarcinoma

Background:Circulating tumor DNA (ctDNA) is a promising biomarker for non-invasive epidermal growth factor receptor mutations (EGFRm) detection in lung cancer patients, but existing methods have limitations in sensitivity and availability. In this study, we used the ΔCt value (mutant cycle threshold [Ct] value–internal control Ct value) generated during the polymerase chain reaction (PCR) assay to convert super-amplification-refractory mutation system (superARMS) from a qualitative method to a semi-quantitative method named reformed-superARMS (R-superARMS), and evaluated its performance in detecting EGFRm in plasma ctDNA in patients with advanced lung adenocarcinoma.Methods:A total of 41 pairs of tissues and plasma samples were obtained from lung adenocarcinoma patients who had known EGFRm in tumor tissue and were previously untreated. EGFRm in ctDNA was identified by using superARMS. Through making use of ΔCt value generated during the detection process of superARMS, we indirectly transform this qualitative detection method into a semi-quantitative PCR detection method, named R-superARMS. Both qualitative and quantitative analyses of the data were performed. Kaplan–Meier analysis was performed to estimate the progression-free survival (PFS) and overall survival (OS). Fisher exact test was used for categorical variables.Results:The concordance rate of EGFRm in tumor tissues and matched plasma samples was 68.3% (28/41). At baseline, EGFRm-positive patients were divided into two groups according to the cut-off ΔCt value of EGFRm set at 8.11. A significant difference in the median OS (mOS) between the two groups was observed (EGFRm ΔCt ≤8.11 vs. >8.11: not reached vs. 11.0 months; log-rank P = 0.024). Patients were divided into mutation clearance (MC) group and mutation incomplete clearance (MIC) group according to whether the ΔCt value of EGFRm test turned negative after 1 month of treatment. We found that there was also a significant difference in mOS (not reached vs. 10.4 months; log-rank P = 0.021) between MC group and MIC group. Although there was no significant difference in PFS between the two groups, the two curves were separated and the PFS of MC group tended to be higher than the MIC group (not reached vs. 27.5 months; log-rank P = 0.088). Furthermore, EGFRm-positive patients were divided into two groups according to the cut-off of the changes in ΔCt value of EGFRm after 1 month of treatment, which was set at 4.89. A significant difference in the mOS between the two groups was observed (change value of ΔCt >4.89 vs. ≤4.89: not reached vs. 11.0 months; log-rank P = 0.014).Conclusions:Detecting EGFRm in ctDNA using R-superARMS can identify patients who are more likely sensitive to targeted therapy, reflect the molecular load of patients, and predict the therapeutic efficacy and clinical outcomes of patients.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

Status and Prospects of PCR Detection Methods for Diagnosing Pathogenic Escherichia coli : A Review

Escherichia coli are the predominant facultative bacteria found in the gastrointestinal tract of animals and humans. Some strains of E. coli that acquire virulence factors and cause foodborne and waterborne diseases in humans are called pathogenic E. coli and can be divided into five pathotypes according to the virulence mechanism: EAEC, EHEC, EIEC, EPEC, and ETEC. Although selective media have been developed to detect E. coli, distinguishing pathogenic strains from non-pathogenic ones is difficult because of their similar biochemical properties. Therefore, it is very important to find a new and effective diagnostic method to identify pathogenic E. coli. With recent advances in molecular biology and whole genome sequencing, the use of polymerase chain reaction (PCR) is increasing rapidly. In this review paper, we provide an overview of pathogenic E. coli and present a review on PCR detection methods that can be used to diagnose pathogenic E. coli. In addition, the possibility of real-time PCR incorporating IAC is introduced. Consequently, this review paper will contribute to solving the current challenges related to the detection of pathogenic E. coli.

PCR technology for screening genetically modified soybeans

Genetically modified (GM) crops involve transferring a specific stretch of DNA into the plant’s genome and giving it new or different characteristics. Since the first industrial application of transgenic crops in the United States in 1996, GM crops are becoming increasingly common in the market. At present, not all crops in the Chinese market are clearly marked as to whether they are GM crops or not. In order to protect consumer’s option to select their preferred products, we decided to conduct experiments to identify soybeans on the market. We hypothesized that soybeans on the market with unknown origin were GM soybeans. Then we established a detecting method using genetic technology and screened the soybeans. In this study, we used GTS 40-3-2 as a positive control, which contains genomic DNA, foreign DNA enolpyruvylshikimate-3-phosphate synthase (EPSPS) and other exogenous elements, and GTS 40-3-2 SOYA BEAN (blank) as negative control, which contains genomic DNA only. To amplify the foreign DNA, we optimized the polymerase chain reaction (PCR) procedure by attempting different annealing temperatures to establish the PCR detection method. According to the electrophoresis results, in the samples from a local store, only genomic DNA lectin could be amplified, which indicated the samples were non-GM soybeans. Our study not only provides a detection method but also lays a foundation for testing soybean and processed soybean products of unknown origin.

Performance of Eastern Mediterranean Region laboratories in the World Health Organization external quality assessment programme for arbovirus diagnostics.

Arboviruses such as dengue virus, yellow fever virus, Zika virus and chikungunya virus are major threats to human health globally, including countries in the Eastern Mediterranean Region (EMR). This study aimed to assess laboratory proficiency in EMR countries for detection of dengue virus, yellow fever virus, Zika virus and chikungunya virus. A global external quality assessment programme for arbovirus diagnostics was developed and run in 2016 and 2018. National-level public health laboratories were instructed to apply the polymerase chain reaction detection method on specimen panels containing dengue virus, yellow fever virus, Zika virus and chikungunya virus. Over both rounds of the programme, 100% of participating EMR laboratories correctly detected yellow fever virus and chikungunya virus, ≥ 84.6% detected dengue fever virus and ≥ 76.9% detected Zika virus. While participating EMR countries demonstrated good proficiency in detecting arboviruses, only half of them were enrolled in the global external quality assessment programme, providing an incomplete picture of regional capacity. Effort should be put into increasing participation in subsequent rounds.

PCR detection for syphilis diagnosis: Status and prospects.

Syphilis, a re‐emerging public health problem worldwide caused by Treponema pallidum subsp pallidum (T. pallidum), usually induces systemic and chronic inflammation in hosts who do not receive timely therapy after exposing to high‐risk factors such as leprous sexual contact. Before the treatment, rapid and accurate detection of syphilis is essential. However, the existing detection methods, which focus on the treponemal or non‐treponemal antibody test, both have inherent limitations. For instance, both of them cannot distinguish the stage and severity of syphilis. Non‐treponemal test such as RPR, which is generally deemed to be used for assessing treatment response, is influenced by biological false positives. Therefore, it is imperative to seek out a new and effective diagnostic test. With recent advancements in molecular biology and whole‐genome sequencing, the molecular diagnosis has increased in popularity, especially the use of polymerase chain reaction (PCR). Here, we firstly present a mini‐review on the research of PCR detection methods used for syphilis diagnosis over the past decade, and we then compare these methodologies to assess their potential and the challenges faced. This information can provide a fresh perspective to help researchers address the current challenges.

Identification of chickpea (Cicer arietinum) in foods using a novel real-time polymerase chain reaction detection method

A novel real-time polymerase chain reaction (PCR) method for detecting chickpea was developed. From homologous sequences of 9-cis-epoxycarotenoid dioxygenase gene (NCED) among leguminous species, chickpea’s NCED was cloned, and the Southern-blot analysis showed that NCED is a single copy gene in a haploid genome. Its coding sequences at the 5′ terminus were found unique to chickpea and conserved among various chickpea varieties. Developed real-time PCR method targeting the unique sequences was specific to chickpea and had detection limit of approximately 30 copies per a reaction, and applicable for qualitative detection of chickpea in various forms of food products including dried, powdered, retort-packed, canned, fermented and pasted. Our results showed that the developed method enables identification of chickpea in foods.

Monitoring the seasonal dynamics of microalgae in the South Sea of Korea by use of a cytochrome c oxidase I DNA barcode

Microalgae are important primary producers in the marine environment, and are also important sources of health foods and medical products. Rapidly growing microalgae have potential use for the production of commercial products, but can also cause harmful microalgal blooms in natural ecosystems. There are many available techniques for the identification of microalgae in natural ecosystems. We used the mitochondrial cytochrome c oxidase subunit I gene as DNA barcode to identify 14 species of microalgae from the South Sea of Korea and to discriminate among similar biogeographic subgroups within species. In addition, we designed species-specific primers targeting the cytochrome c oxidase subunit I gene to evaluate monthly changes of microalgae throughout the year. Chaetoceros brevis, Asterionellopsis glacialis and Stephanopyxis turris were present during all seasons, whereas Skeletonema japonicum, Nitzschia improvisa, Ditylum brightwellii and Chaetoceros diadema were only detected during winter and spring. Our results indicate that species-specific polymerase chain reaction of the cytochrome c oxidase subunit I gene can be used to monitor the seasonal dynamics of microalgae in the South Sea of Korea. This polymerase chain reaction detection method successfully identified the 14 most common species of microalgae with the same polymerase chain reaction condition near Tongyeong, in the South Sea of Korea.

Bacterial colonisation of suture material after routine neurosurgical procedures: relevance for wound infection.

Wound healing impairment is a serious problem in surgical disciplines which may be associated with chronic morbidity, increased cost and patient discomfort. Here we aimed to investigate the relevance of bacterial colonisation on suture material using polymerase chain reaction (PCR) to detect and taxonomically classify bacterial DNA in patients with and without wound healing problems after routine neurosurgical procedures. Repeat surgery was performed in 25 patients with wound healing impairment and in 38 patients with well-healed wounds. To determine the presence of bacteria, a 16S rDNA-based PCR detection method was applied. Fragments of 500bp were amplified using universal primers which target hypervariable regions within the bacterial 16S rRNA gene. Amplicons were separated from each other by single-strand conformation polymorphism (SSCP) analysis, and finally classified using Sanger sequencing. PCR/SSCP detected DNA of various bacteria species on suture material in 10/38 patients with well-healed wounds and in 12/25 patients with wound healing impairment including Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes and Escherichia coli. Microbiological cultures showed bacterial growth in almost all patients with wound healing impairment and positive results in PCR/SSCP (10/12), while this was the case in only one patient with a well-healed wound (1/10). Colonisation of suture material with bacteria occurs in a relevant portion of patients with and without wound healing impairment after routine neurosurgical procedures. Suture material may provide a nidus for bacteria and subsequent biofilm formation. Most likely, however, such colonisation of sutures is not a general primer for subsequent wound infection.

Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.

An attempt to develop a detection method specific for virulent duck plague virus strains based on the UL2 gene B fragment

ABSTRACTA comparison of the unique long region 2 (UL2) gene sequences between 10 virulent and 11 attenuated duck plague virus (DPV) strains (including all DPV UL2 gene sequences registered in GenBank) showed that the UL2 genes in the attenuated DPV strains had a 528 bp deletion in the B fragment. Primers were designed based on the B fragment sequence of the UL2 gene in an attempt to establish a fluorescence quantitative polymerase chain reaction (PCR) and a conventional PCR detection method that could specifically detect virulent DPV strains (i.e. positive detection for virulent DPV strains and negative detection for attenuated DPV strains). Additionally, PCR products were cloned for sequence analysis. These two methods detected five attenuated DPV strains in addition to the virulent DPV strains. Sequence analysis of the PCR products showed that the amplified products were the B fragments of the UL2 gene. These results indicated that detection methods specific for virulent DPV strains could not be established using primers designed based on the UL2 gene B fragment.

Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method

Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities.

Inconclusive role of human papillomavirus infection in breast cancer.

BackgroundEpidemiological studies have examined the association between human papillomavirus (HPV) and breast cancer, but the findings are inconclusive. This study aimed to detect the prevalence of HPV in breast cancer tissue in patients from northeastern China and define the association between HPV and breast cancer using meta-analysis.MethodsPolymerase chain reaction (PCR) was used to test cutaneous or mucosal HPV DNA sequence in 77 breast cancer samples and 77 corresponding adjacent normal tissues. The prevalence of HPV in breast cancer was estimated by pooling data from 38 studies. A meta-analysis of 16 case–control studies was conducted to investigate the association between HPV and breast cancer.ResultsWe did not find HPV DNA sequence in any of the 154 tissue specimens we tested. However, our meta-analysis revealed a HPV prevalence of 30.30 % (95 % confidence interval [CI] = 22.30–38.40 %) among breast cancer cases; most of these involved high-risk HPV types (35.50 %, 95 % CI = 25.00–46.10 %). HPV prevalence in breast cancer varied by geographic region, publication period, and PCR detection method. An increased risk of breast cancer was observed in association with exposure to HPV (odds ratio [OR] = 3.24, 95 % CI = 1.59–6.57), which was influenced by geographic region, HPV DNA source, PCR primer used, and publication period.ConclusionsHPV, especially high-risk HPV types, may be associated with an increased risk of breast cancer, and this association varies dramatically among geographic regions.