Polymerase Chain Reaction Procedure Research Articles

Manually operated microtube automatic capper/decapper system for clinical laboratory and biological laboratory personnel

Polymerase chain reaction (PCR) is an effective method for diagnosing infectious diseases and has been the primary method throughout the novel coronavirus disease (COVID-19) pandemic. PCR tests (from specimen collection to result acquisition) involve sample pretreatment, nucleic acid extraction, and PCR procedure. Automating the pretreatment process is crucial to mitigate the risk of infection for workers and to reduce the likelihood of sample contamination-triggered misdiagnosis, particularly when handling centrifuge tubes, cryopreservation tubes, and microtubes. Robotic systems have been engineered to automate cell culture and PCR-based diagnosis, predominantly designed for use with screw-capped containers. However, this leaves a notable gap in automation solutions for microtubes equipped with press-type caps. To address this gap, we developed a versatile microtube capper/decapper system. On the other hand, many tasks of manual operation using microtubes, which are routinely conducted in clinical tests and biological experiments, are performed. Compared to screw-type caps for centrifuge and cryopreservation tubes, press-type caps for microtubes present a considerably higher risk of the worker's fingers contacting the inside of the cap and/or generating airborne droplets. Despite the risks of contamination and infection derived from the manual handling of microtube caps, which can compromise diagnosis/experiment accuracy and worker safety, devices for manually opening and closing microtube caps without direct contact remain lacking. Therefore, leveraging the technology from the developed versatile microtube capper/decapper system for laboratory automation, we created a manually operated microtube equipped with an automatic capper/decapper system tailored for personnel in clinical and biological laboratories.In this study, we first examined the required specifications and prerequisites for a manual microtube capper/decapper and clarified the operating methods, operating procedures, operation environment, device size, accompanying functions, etc. Based on the required specifications and preconditions, we proceeded with the mechanical and control design of the conceptual model, manufactured a prototype, and confirmed its basic functions and performance. The compliant to the required specifications and preconditions and the usefulness of the proposed manual microtube capper/decapper were validated through various experiments and demonstrations. Using the proposed microtube capper/decapper, even small-scale operations, which are challenging to streamline, can be performed nearly as efficiently as full manual operations. Although operation time was not reduced, the ability to open and close microtubes without manual contact is crucial for improving diagnostic and experimental accuracy and for reducing the burden on and enhancing the safety of laboratory personnel. Because microtubes are used in various clinical tests and biological experiments, we believe that the proposed system can markedly reduce the workload for personnel across numerous clinical and biological laboratories.

PCR virtual temperature sensor design based on system modeling and identification

The reaction results of Polymerase chain reaction (PCR) are highly sensitive to temperature accuracy, but the reaction liquid temperature is hard to be measured directly. In this paper, a virtual temperature sensor is creatively designed to directly obtain the reaction liquid temperature using a model-switching approach. This study first establishes the mathematical models of the temperature control system and obtains precise system models for the heating and cooling stages. Based on this, a virtual sensor is constructed, and a closed-loop fuzzy PID control system is established. Furthermore, the accuracy of the designed virtual temperature sensor is verified through simulation and experimentation. Simulation results show that the fuzzy PID control method has higher temperature accuracy, with a deviation of 0.23 °C, making it more suitable for Peltier systems, as Peltier systems suffer from inconsistent temperature rise and fall models. The experimental results show that the control method using the virtual sensor achieves higher precision in liquid temperature control, with an accuracy of ±0.21 °C. Finally, standard PCR procedures are performed for designed chip, and the results indicate that the temperature sensor and control system designed in this study exhibit significantly higher amplification efficiency, approximately 16 times greater than traditional control methods.

Molecular detection and association of 12.1kb deletion within the high mobility AT-hook 2 gene in the Netherlands dwarf rabbit (Oryctolagus Cuniculus).

Rabbits are mainly bred for human consumption and medical research. However, it has been recently showed that several rabbit breeds are also kept as pets for human leisure. The Netherlands dwarf rabbit is currently in the immense interest of many Vietnamese customers due to its personality and miniature stature. However, 12.1kb deletion from position 44,709,089 to 44,721,236bp in the high mobility AT-hook 2 (HMGA2) gene on chromosome 4 was identified as the structural variant causing dwarfism and altered craniofacial development in this breed. It has been documented that HMGA2 plays an important role in regulating growth and individuals with genotype HMGA2 del/del are fatal several days after birth. Despite the economically high value of the Netherlands dwarf rabbit, there has been no study on the genetic survey of lethal alleles in this breed in Vietnam. The aim of this study is to develop a fast and reliable method to screen the frequency of lethal alleles of HMGA2 in the South of Vietnam. Rabbit saliva was collected, and DNA extraction was followed. Multiplex polymerase chain reaction (PCR) with three primers was optimized and performed to detect the presence of 12.1kb deletion within the HMGA2 sequence. Our data showed that the 12.1kb deletion in the Netherlands dwarf rabbit population was detected by our optimized multiplex PCR. In 100 rabbit animals, 34 and 16 individuals were homozygous wild type (+/+) and homozygous mutant (del/del), respectively, while 50 rabbits were heterozygous. The frequency of HMGA2 lethal allele carrier was 66% (66/100 individuals). Our results indicated that we successfully developed a fast, accurate multiplex PCR to detect carrier individuals. Verification of the genotypes was followed by sequencing. We recommend implementing our multiplex PCR procedure in genetic selection for carrier and homozygous wild-type animals in the mating scheme to prevent the lethality of the rabbit offspring. Additionally, awareness should be raised among rabbit breeders to monitor the genetic makeup of the Netherlands dwarf rabbit populations. However, due to the limitation of the sample size, more samples should be taken in future studies to obtain the genetic frequency of the HMGA2 lethal allele more accurately.

Polyketide synthase positive Escherichia coli one-time measurement in stool is not informative of colorectal cancer risk in a screening setting.

Environmental factors like the pathogenicity island polyketide synthase positive (pks+) Escherichia coli (E. coli) could have potential for risk stratification in colorectal cancer (CRC) screening. The association between pks+ E. coli measured in fecal immunochemical test (FIT) samples and the detection of advanced neoplasia (AN) at colonoscopy was investigated. Biobanked FIT samples were analyzed for both presence of E. coli and pks+ E. coli and correlated with colonoscopy findings; 5020 CRC screening participants were included. Controls were participants in which no relevant lesion was detected because of FIT-negative results (cut-off ≥15 μg Hb/g feces), a negative colonoscopy, or a colonoscopy during which only a nonadvanced polyp was detected. Cases were participants with AN [CRC, advanced adenoma (AA), or advanced serrated polyp (ASP)]. Existing DNA isolation and quantitative polymerase chain reaction (qPCR) procedures were used for the detection of E. coli and pks+ E. coli in stool. A total of 4542 (90.2%) individuals were E. coli positive, and 1322 (26.2%) were pks+ E. coli positive. The prevalence of E. coli in FIT samples from individuals with AN was 92.9% compared to 89.7% in FIT samples of controls (p = 0.010). The prevalence of pks+ E. coli in FIT samples from individuals with AN (28.6%) and controls (25.9%) was not significantly different (p = 0.13). The prevalences of pks+ E. coli in FIT samples from individuals with CRC, AA, or ASP were 29.6%, 28.3%, and 32.1%, respectively. In conclusion, the prevalence of pks+ E. coli in a screening population was 26.2% and did not differ significantly between individuals with AN and controls. These findings disqualify the straightforward option of using a snapshot measurement of pks+ E. coli in FIT samples as a stratification biomarker for CRC risk. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Prevalence of CYP2C19*2 and CYP2C19*3 Allelic Variants and Clopidogrel Use in Patients with Cardiovascular Disease in Trinidad & Tobago.

Trinidad & Tobago has the highest prevalence of cardiovascular disease (CVD) in the Caribbean and clopidogrel is a ubiquitously used treatment. Yet, the extent of genetically mediated clopidogrel resistance is unknown. To determine this, we investigated whether the association between CYP2C19*2 and CYP2C19*3 genetic variants and clopidogrel resistance holds, and calculated the frequencies of these in the Trinidadian CVD population. Demographic data, clinical data, and a saliva sample were collected under informed consent from 22 patients with CVD on dual anti-platelet therapy whose biochemical resistance to clopidogrel is known, and a further 162 patients accessing the main public CVD clinic in Trinidad and who are either currently being treated or are likely to be treated with clopidogrel. A polymerase chain reaction (PCR) and restriction enzyme digestion procedure was used to genotype each patient for the CYP2C19*2 and CYP2C19*3 allelic variants. Genotype was compared to known clopidogrel resistance in the 22 patients, and to disease status and clopidogrel usage in the larger cohort. CYP2C19*2 genotype was concordant with clopidogrel resistance. CYP2C19*2 was detected in 61.1% (99/162) of patients and CYP2C19*3 was undetected. Clopidogrel was the most prescribed antiplatelet therapy (42%). A total of 120 people presented with coronary artery disease (CAD) and 52.5% of these (n = 63/120) are currently prescribed clopidogrel. 63.5% (40/63) of patientswith CAD who are prescribed clopidogrel carry the CYP2C19*2 allele; ten homozygous and 30 heterozygous. Indian patients comprised 65% of the cohort and were four times more likely to carry the CYP2C19*2 allele than African patients. A large proportion of Trinidadian patients with CVD who are prescribed or may be prescribed clopidogrel carry genetic variants associated with clopidogrel resistance. These results emphasize the clinical need for further investigation into whether CYP2C19*2 genotype should guide clopidogrel use for the cardiovascular disease population in Trinidad & Tobago. A slide deck is available for this article.

Human papillomavirus DNA testing with the urine sample is not yet available: the accuracy of two distinct kits.

The aim of this study was to assess the results and efficiency of two real-time polymerase chain reaction procedures for detecting human papillomavirus utilizing urine samples. This study comprised 151 patients who had previously tested positive for human papillomavirus in their cervical samples. Two different commercial real-time polymerase chain reaction techniques were used for identification and genotyping human papillomavirus in urine specimens. The urine samples of 151 patients were evaluated via the Roche Cobas test, and the urine samples of 91 patients were also evaluated via the Qiagen test. The overall consistency of urine and cervical swab specimens for the identification of human papillomavirus in Roche Cobas and Qiagen tests were 44.8 and 44%, respectively. The rates of positive human papillomavirus results from urine samples were 57 and 70.3%, respectively. The overall concordance among Roche Cobas and Qiagen tests utilizing urine samples for human papillomavirus type 16/18 was 84.3% with a kappa value of 0.675, and for other high-risk-human papillomavirus, it was 75.60% with a kappa value of 0.535. Roche Cobas showed high concordance with Qiagen test. human papillomavirus positivity was not detected in all urine samples. It is still inappropriate to recommend the use of urine liquid biopsy for the accurate and reliable detection of human papillomavirus. Due to the lack of a standardized tool, the utilization of urine samples as a screening human papillomavirus test remains a challenge.

Screening for SARS-CoV-2 infection in women prepared for elective Cesarean Section: A tertiary hospital study

Background and objectives: The SARS-CoV-2 infection is a major concern as universal testing should be undertaken to pregnant women due to the high danger of transmission from a pregnant woman to her newborn. This study was conducted to report the rate of SARS-CoV-2 infection in women admitted to hospital for purpose of preparation for elective cesarean section and to find out their risk factors and any histories of the infection previously. Methods: A cross sectional study was conducted on 1000 on late third trimester pregnant women whom were prepared for elective cesarean section at department of Obstetrics and Gynecology, Erbil-Iraq from 1st Aug 2021 till 30th Jul 2022, History of previous SARS-CoV-2 infection and current signs and symptoms of the infection were recorded. All women underwent SARS-CoV-2 infection screening using nasal and throat swabs using polymerase chain reaction procedure. Results: Only (21.2%) of them were positive for the virus. There was a significant association between the current confirmed polymerase chain test with education, being a smoker, and all known signs and symptoms of the infection, such as fever, cough, and shortness of breath. When the clinical and social characteristics of women with low-risk and high-risk pregnancies were compared, there was a strong link between education, smoking, and loss of taste. Conclusion: Pregnant women may be advised to be tested for SARS-CoV-2 before cesarean section as almost one-quarter of participant were positive for the infection regardless of having sign and symptoms or not.

Identification of Candida Isolates from Cancer Patients Using Multiplex PCR

Aim of this study was to identify the Candida isolates from Cancer Hospital Maharagama, Sri Lanka by using Multiplex PCR (polymerase chain reaction) method. In this study, Multiplex PCR was used to identify Candida isolates from cancer patients at Apeksha Hospital, Sri Lanka. In multiplex PCR, 18 specific primers (nine primer pairs) were separated into three groups for multiplex PCR (PS I, PS II and PS III). Different sized PCR products corresponding to each Candida species were produced by PCR using the primer mixes. Among 52 Candida isolates, 49 were identified using the multiplex PCR procedure. Candida tropicalis was found to be the most prevalent species (38%), followed by Candida parapsilosis (31%), Candida albicans (13%), Candida glabrata (8%) and Candida krusei (4%). The study concludes that multiplex PCR is a better approach for the identification of Candida species and recommends the use of it for clinical and diagnostic purposes and for research purposes.

SEX EFFECT ON THE TELOMER LENGTH, ELEMENTAL COMPOSITION, AND PHYTOCHEMICAL CONTENT OF HIGHLAND PAPAYA (VASCONCELLEA PUBESCENS A.DC.) LEAVES

Highland papaya (Vasconcellea pubescens A.DC.) is a monoecious and dioecious plant with female and male organs. In Indonesia, three sexes of highland papaya exist with different telomere lengths, which protect chromosomes and deoxyribonucleic acid (DNA) from damage. Therefore, the purposeful study aimed to determine the telomere length, elemental composition, and phytochemical content of highland papaya leaves belonging to their different sexes. The telomere length observed and studied in said leaves (female, male, and monoecious) used the polymerase chain reaction (PCR) procedure. A scanning electron microscopy (SEM) analysis provided a more detailed material of leaves, while an energy dispersive X-Ray (EDX) helped observe elemental composition. Phytochemical content analysis ran by histochemical analysis. The results showed that the telomere lengths of young female, male, and monoecious leaves differed at 381, 391, and 396.66, respectively. According to the SEM analysis, nonsignificant differences occurred in the leaf surface of the three sexes of highland papaya leaves. Observation with EDX showed that sodium (1.98%) surfaced in female plant leaves; however, sodium and chlorine did not show in male plants. Monoecious leaves had sodium and chlorine at 0.88% and 0.28%, respectively. The histochemical analysis provided an overview of the distribution of flavonoids and tannins in young leaves of highland papaya. A discovery also noted the existence of both compounds in the adaxial and abaxial epidermis, mesophyll, xylem, phloem, sheath parenchyma, secretory cavities, and trichomes. The study concluded that sex affects telomere length, elemental composition, and the phytochemical content of highland papaya leaves. The study suggests that knowing the highland papaya’s sex is vital in plant breeding and genetics and could help improve plant health and productivity.

Serotype replacement and an increase in non-encapsulated isolates among community-acquired infections of Streptococcus pneumoniae during post-vaccine era in Japan

ObjectivesIt is feared that the serotype replacement of Streptococcus pneumoniae occurred by the introduction of pneumococcal vaccines as periodical inoculation leads to reduced efficacy of the approved vaccines and altered antimicrobial susceptibility. MethodsWe determined serotypes of 351 S. pneumoniae isolates collected at a commercial clinical laboratory in Hokkaido prefecture, Japan, from December 2018 to February 2019 by using the polymerase chain reaction procedure of the US Centers for Disease Control and Prevention. Antimicrobial susceptibility and resistance gene profiles were also examined. ResultsVaccine coverage rates were 7.9% for 13-valent conjugate vaccine, and 32.5% for 23-valent polysaccharide vaccine, respectively. Non-typable strains were 19.7%. cpsA-positive isolates (group I), and null capsule clade (NCC)1, NCC2 and NCC3 (group II) comprised 31.3%, 28.4%, 32.8%, and 7.5% of the 69 non-typable strains, respectively. No penicillin-resistant/intermediate isolates were found; however, serotypes 35B and 15A/F showed low susceptibility to β-lactams. Only five strains (1.4%) were levofloxacin-resistant, and all were from the older persons, and three strains were serotype 35B. ConclusionThe progression of serotype replacement in non-invasive pneumococcal infections has occurred during the post-vaccine era in Japan, and non-encapsulated isolates, such as NCC, have increased. Antimicrobial susceptibility is not worsened.

A Genotyping Method for Detecting Foreign Buffalo Material in Mozzarella di Bufala Campana Cheese Using Allele-Specific- and Single-Tube Heminested-Polymerase Chain Reaction.

Mozzarella di Bufala Campana (MdBC) cheese is a Protected Designation of Origin (PDO) product that is important for the economy and cultural heritage of the Campania region. Food fraud can undermine consumers' trust in this dairy product and harm the livelihood of local producers. The current methods for detecting adulteration in MdBC cheese due to the use of buffalo material from foreign countries could exhibit limitations associated with the required use of expensive equipment, time-consuming procedures, and specialized personnel. To address these limits here, we propose a rapid, reliable, and cost-effective genotyping method that can detect foreign buffalo milk in a counterpart from the PDO area and in MdBC cheese, ensuring the quality and authenticity of the latter dairy product. This method is based on dedicated allele-specific and single-tube heminested polymerase chain reaction procedures. By using allele-specific primers that are designed to detect the nucleotide g.472G>C mutation of the CSN1S1Bbt allele, we distinguished an amplicon of 330 bp in the amplification product of DNA when extracted from milk and cheese, which is specific to the material originating from foreign countries. By spiking foreign milk samples with known amounts of the counterpart from the PDO area, the sensitivity of this assay was determined to be 0.01% v/v foreign to PDO milk. Based on a rough estimate of its simplicity, reliability, and cost, this method could be a valuable tool for identifying adulterated buffalo PDO dairy products.

A Simple, Rapid, and Cost-Effective PCR Procedure for Detection of NUDT15 Gene Variants in Vietnamese Patients with Acute Lymphoblastic Leukemia.

Objective The NUDT15 variants impact thiopurine dose selection in acute lymphoblastic leukemia patients. The ability to rapidly detect variants is important in clinical practice. This study aims to develop a simple polymerase chain reaction (PCR) procedure for detecting NUDT15 variants in Vietnamese patients. Materials and Methods Sanger sequencing was used to determine NUDT15 variants from 200 patients. We designed primers and optimized the PCR procedure for detection of wild-type and variant alleles and compared with Sanger sequencing results. Results The inserted variant c.55_56insGAGTCG was detected by differences in size through conventional PCR. The tetra-primer amplification refractory mutation system PCR was successful in detecting two variations, c.52G > A and c.415C > T. The sensitivity and specificity of PCR procedure achieved 100% when compared to 200 Sanger sequencing results. Conclusion Our PCR procedure is suitable for replacing Sanger sequencing to detect the NUDT15 variants in clinical setting.

The Coronavirus Calendar (CoronaCal): a simplified SARS-CoV-2 test system for sampling and retrospective analysis.

To develop a biological diary (CoronaCal) that allows anyone in the community to collect and store serial saliva samples and chart symptoms on ordinary printer paper. Diaries were analyzed for the presence of SARS-CoV-2 RNA using established polymerase chain reaction (PCR) procedures. CoronaCal diaries were distributed to volunteer subjects in the community during the peak of the COVID-19 outbreak in New York. Volunteers collected their own daily saliva samples and self-reported symptoms. SARS-CoV-2 RNA extracted from CoronaCals was measured using qPCR and RNA levels were correlated with reported symptoms. SARS-CoV-2 RNA was detected in CoronaCals from nine of nine people with COVID-19 symptoms or exposure to someone with COVID-19, and not in one asymptomatic person. CoronaCals were stored for up to 70 days at room temperature during collection and then frozen for up to four months before analysis, suggesting that SARS-CoV-2 RNA is stable once dried onto paper. Sampling saliva on simple paper provides a useful method to study the natural history and epidemiology of COVID-19. The CoronaCal collection and testing method is easy to implement, inexpensive, non-invasive and scalable. The approach can inform the historical and epidemiological understanding of infections in individuals and populations.

Detection of AcrA and AcrB Efflux Pumps in Multidrug-Resistant Klebsiella pneumonia that Isolated from Wounds Infection Patients in Al-Diwaniyah Province.

Many infections produced by multidrug-resistant (MDR) Klebsiella pneumoniae are the main cause of death and treatment restrictions worldwide. In K. pneumoniae, the efflux pump system is dangerous in drug resistance. Therefore, this study was designed to investigate the involvement of the AcrA and AcrB efflux pumps in antibiotic resistance in Klebsiella pneumoniae isolated from wound patients. During June 2021-February 2022, 87 clinical isolates of Klebsiella pneumonia bacteria were obtained from wound samples patients consulted to the hospitals in AL-Diwaniyah province, Iraq. The disc diffusion method performed an antibiotic susceptibility test after microbiological/biochemical identification. The polymerase chain reaction (PCR) technique was used to examine efflux genes' prevalence (acrA and acrB). The results showed that resistance to Carbenicillin 72 (82.7%), Erythromycin 66 (75.8%), Rifampin 58 (66.6%), Ceftazidime 52 (59.7%), Cefotaxime 44 (50.5%), Novobiocin 38 (43.6%), Tetracycline 32 (36.7%), Ciprofloxacin 22 (25.2%), Gentamicin 16 (18.3%), Nitrofurantoin 6 (10.3%) in Klebsiella pneumoniae isolates. The PCR procedure revealed that the occurrence of the acrA and acrB genes is 55 (100%) and 55 (100%), respectively. The findings of this investigation show that the AcrA and AcrB efflux pumps play a crucial character in antibiotic resistance in multidrug-resistant Klebsiella pneumoniae bacterial isolates. As a result of the unintentional transmission of antimicrobial resistance genes, precise detection of resistance genes using molecular approaches is required to switch the extent of resistant strains.

Investigation of mobile colistin resistance (mcr) genes among carbapenem resistance Pseudomonas aeruginosa isolates from bovine mastitis in Mashhad, Iran

BackgroundThe most severe pathology affecting the dairy industry is bovine mastitis (BVM). Pseudomonas aeruginosa is widely associated with several infections, including the BVM. The current study aimed to investigate the incidence of carbapenemase and mobile-colistin resistance (mcr) genes among resistant P. aeruginosa isolates from Mashhad, northeast Iran. MethodsThe current study was conducted on archived P. aeruginosa isolates from mastitis. The antimicrobial susceptibility testing was performed; the carbapenem resistance isolates were subsequently sought to demonstrate the carbapenemase-producing isolates using the modified-carbapenem inactivation method. In addition, the colistin broth disc elution method was carried out to determine the frequency of resistance against colistin. Finally, resistance isolates were further tested for detecting related carbapenemase and mobile-colistin resistance (mcr) genes, including blaNDM, blaOXA-48, blaVIM, blaKPC, blaIMP, and mcr1-5, using the polymerase chain reaction procedure (PCR). ResultsAmong carbapenem-tested antibiotics, meropenem has revealed the least activity (8 %); on the other hand, aztreonam, gentamycin, and tobramycin were 100 % effective. Two isolates (4 %) have shown colistin resistance (with MIC ≥4 μg/mL). The PCR analysis has displayed the high frequency of blaVIM among (43/43) meropenem resistance isolates. Also, the presence of mcr-2 was detected for two colistin resistance isolates. ConclusionThe substantial resistance to carbapenems and emerging colistin resistance in veterinary have been observed. Thus, the following antimicrobial stewardship in farm animals reduces the increasing multi-drug resistance of P. aeruginosa isolates.

Distribution of 22 Single Nucleotide Polymorphisms in 13 Cytokine Genes in Malays, Chinese, and Indians in Peninsular Malaysia.

Introduction: Cytokines are cell signaling glycoproteins that are particularly important in immunity and inflammatory responses. Therefore, variations, such as single nucleotide polymorphisms (SNPs), in genes encoding for cytokines may have important consequences for their roles in health. Materials and Methods: A total of 222 unrelated, healthy, and un-admixed Malays (n = 97), Chinese (n = 77), and Indians (n = 48) with a median age of 30 years old (range 21-50) were typed for 22 cytokine gene SNPs: IL-1α -889 T/C, IL-1β (-511 T/C, +3962 T/C), IL-1R pst1 1970 T/C, IL-1RA mspa1 11100 T/C, IL-4Rα +1902 G/A, IL-12 - 1188 C/A, IFN-γ +874 A/T, TGF-β (cdn 10 C/T, cdn 25 G/C), TNF-α (-308 A/G, -238 A/G) IL-2 (+166 G/T, -330 T/G), IL-4 (-1098 T/G, -590 T/C, -33 T/C), IL-6 (-174 C/G, nt565 G/A), and IL-10 (-1082 G/A, -819 C/T, -592 A/C). This involved using well-established polymerase chain reaction procedures with sequence-specific primers and restriction fragment length polymorphism methods. Results: The majority of the screened cytokine gene SNPs are polymorphic in all three ethnicities. Exceptions include TGF-β cdn 25 (G/C), IL-1β +3962 (T/C), and TNF-α -238 (A/G), which were all observed to be monomorphic in Malays, Chinese and Indians. Many of the analyzed cytokine gene SNP genotypes deviated from Hardy-Weinberg equilibrium and the three ethnic study groups were all well-separated from reference Asian, African and European populations in a principal component analysis plot. Conclusion: We successfully typed 22 SNPs in 13 cytokine genes from genetic material collected from unrelated and un-admixed Malay, Chinese and Indian individuals in Peninsular Malaysia. These new cytokine gene population datasets reveal interesting contrasts with other populations.

Differentiating individuals of Armillaria species in New Zealand forests

Background: Armillaria novae-zelandiae and A. limonea occur naturally as wood decay fungi in native forests in New Zealand. As pathogens they are responsible for significant root disease in trees and shrubs in plantations, crops and urban parks and gardens. A thorough understanding of their population dynamics entails knowledge of the spatial arrangement of their individual mycelia or genets. In previous work the distributions of vegetative compatibility groups (VCGs) of these fungi were mapped in an area of native forest prior to and after replacement by a young Pinus radiata plantation. With the advent of molecular technology, it has become possible to test species identities made earlier using culture techniques and to verify whether or not their VCGs, determined by incompatibility reactions between paired cultures, represent distinct individual genets. Methods: Stock subcultures of isolates representing each VCG were recovered from storage in order to obtain DNA. Extracted DNA was subjected to a polymerase chain reaction procedure (UP-PCR) using 11 universal primers to assess genetic variation between subcultures. Bands were scored as either present or absent for each primer-subculture combination and cluster analysis was undertaken by generating dendrogram trees to reveal genetic groupings among subcultures. Results: DNA cluster analysis divided subcultures of isolates into two species groups, A. novae-zelandiae and A. limonea, corresponding to identities determined through culture morphology. Within species, subcultures grouped into clusters that matched VCGs determined by earlier culture pairing. There was little indication of genetic variation within VCGs, except for one of A. limonea, which comprised two sub-clusters. Conclusions: The Armillaria species and VCGs identified by culture techniques in the laboratory were verified by independent molecular methodology. In general, the VCGs represent discrete individual genets or colonies in the field. Techniques that differentiate isolates based on differences in their DNA sequence provide a quick alternative to time-consuming laboratory culture methods for resolving population spatial structure. However, some complementary isolate pairing may be necessary when rationalising the significance of groupings in dendrogram trees.

Effects of trehalose on NFE2L2, catalase, and superoxide dismutase in the kidney of aged rats.

Aging is associated with an increase in oxidative stress, which damages organs such as the kidney. Trehalose has abundant beneficial activities including antioxidative effects. This study aimed to investigate the effects of trehalose on several antioxidant parameters of the aged kidney. Wistar rats were divided into three groups: young (4 months), aged (24 months), and aged-trehalose. The third group was treated with 2% trehalose for one month. The expression of target genes and enzyme activities in the kidney of the animals were evaluated by quantitative polymerase chain reaction (qPCR) and enzyme colorimetric procedures, respectively. Protein levels of NFE2L2 showed a 50% reduction in aged rats compared to young rats (P<0.001), which was restored by trehalose intervention. In addition, the activity and mRNA levels of catalase (CAT) increased in aged rats while treatment with trehalose reversed this trend. On the other hand, superoxide dismutase (SOD) activity was reduced in the kidneys of aged rats but was not affected by trehalose intervention .It is concluded that trehalose supplementation alleviates the antioxidant system impairments in the kidneys of aged rats. However, further investigations are needed to thoroughly describe the antioxidative impacts of trehalose on the kidneys during aging.

Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand.

ABSTRACTLoop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.

High mortality rate of shipping fever cases in cattle caused by bovine herpesvirus type 1 (BoHV-1)

This study reports the high prevalence and molecular characterization of BoHV-1 infection in imported cattle with respiratory system disease after international transport. A high mortality rate of 14.16% (51/360) was reported in a group of animals imported from Hungary to Turkey in 2019. A total of 17 samples were evaluated (3 lung tissue and 14 nasal swab samples) from 15 cattle aged 6 to 9 months not vaccinated against BoHV-1. Virus isolation, polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) procedures were performed within the scope of this study. By virus isolation in MDBK cells, cytopathologic effects was detected in 8 samples (3 lung tissue and 5 nasal swabs samples). The same eight samples were also found positive by BoHV-1 PCR targeting gC (UL44) gene region. According to the sequencing result, the sample (ID: 10054) dropped into a cluster of BoHV-1.1. The REA was applied to the samples to confirm the results of phylogenetic analysis. All of the isolates were identified in the subgroup BoHV-1.1 by REA. These results showed a high mortality risk for imported animals and the possibility for BoHV-1 entering the receiving country via imported animals after transport. This event is a serious problem both for the control of BoHV-1 as well as for animal health and welfare.