DNA Polymerase Chain Reaction Assay Research Articles

A comparison of branched DNA and reverse transcriptase quantitative polymerase chain reaction methodologies for quantitation of lipid nanoparticle encapsulated mRNA.

Messenger RNA (mRNA)-based therapeutics have emerged as a promising modality for various clinical applications, necessitating robust methods for mRNA quantification. This biodistribution study compares the performance of branched DNA and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays for measuring lipid nanoparticle-encapsulated mRNA. Following intravenous administration of nascent peptide imaging luciferase mRNA (1mg/kg) to rats, mRNA levels in various tissues and serum were quantified using both assays. Statistical analyses, including Bland-Altman, Deming regression and Passing-Bablok regression, were employed to assess method comparability and reproducibility. The results indicated that mRNA pharmacokinetics measured by branched DNA and RT-qPCR were largely consistent across tissues, with RT-qPCR showing greater reproducibility across multiple laboratories. RT-qPCR also demonstrated a wider dynamic range and higher sensitivity, making it a more versatile option for large-scale studies. Despite some differences in data due to tissue types and timepoints, both methods provided comparable pharmacokinetic profiles for mRNA quantification. This study underscores the importance of selecting an appropriate quantification method based on study requirements and highlights RT-qPCR's adaptability for multisite research, especially for the clinical development of mRNA-based therapeutics.

Genetic Variability in Leishmaniasis-Causing Leishmania infantum in Humans and Dogs from North-East Spain.

Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*-G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum, demonstrating their suitability for fingerprinting and strain monitoring.

The CD19+ B cell as a marker for the febrile children infected with influenza A and Omicron variant.

H3N2 and Omicron are common pathogens of respiratory infections in children. This study aimed to explore dynamic changes of lymphocyte subsets and the diagnostic value of CD19+ B cell in children infected with influenza A and Omicron. One hundred and sixty-five in-patients with H3N2, 175 in-patients with Omicron variant, and 50 age-matched healthy children from Children's Hospital of Soochow University were included in this study. The participants underwent 13 respiratory pathogens by DNA polymerase chain reaction (PCR), sputum culture, severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) DNA PCR, routine blood, and lymphocyte subset assays within 24 h of admission. The neutrophils, neutrophil-to-lymphocyte ratio, and monocyte-to-lymphocyte ratio in the H3N2 and Omicron groups were significantly higher than in the control groups (p < 0.05). However, the lymphocytes and eosinophils in the H3N2 and Omicron groups were lower than the control groups (p < 0.05). The CD3+ T cell, CD3+ CD4+ T cell, CD3+ CD8+ T cell, CD3- CD19+ B cell, and natural killer cell were lower in the H3N2 and Omicron groups than in the control group (p < 0.05). The CD3- CD19+ cell in the Omicron group was higher than that in the H3N2 group but lower than that in the control group (p < 0.05). In addition, CD3- CD19+ cell had good diagnostic value for H3N2 (area under the receiver operating characteristic curve = 0.902, p < 0.05). The children with H3N2 were more likely to have lower lymphocytes than children with Omicron. Additionally, B-cell count had good diagnostic value for H3N2.

Screening of West Nile Virus, Herpesvirus, and Parvovirus in Rattus spp. in Klang Valley, Malaysia

Free-roaming and scavenging lifestyles of Rattus spp. in densely populated urban areas expose them to multiple pathogens that facilitate the transmission of infection to the human population more rapidly, raising public health concerns. There is limited information on the status of rat susceptibility to virus infection, particularly West Nile virus (WNV), herpesvirus, and parvovirus, to prepare for emerging zoonosis. A total of 23 (n = 23) blood samples collected from Rattus spp. in the wet market areas of Klang Valley, Malaysia, were subjected to molecular assay using a one-step reverse transcription-polymerase chain reaction (RT-PCR) to detect the highly conserved region of the WNV capsid and pre-membrane protein via nested polymerase chain reaction (PCR) assay targeting highly conserved amino acid motifs within the herpesviral DNA-directed DNA polymerase gene (DPOL) and polymerase chain reaction (PCR) assay targeting the parvovirus non-structural (NS) protein. As a result, 4 out of 23 (17.39%) rats were positive for herpesvirus DNA, but none were positive for WNV RNA and parvovirus DNA. The positive PCR amplicons of herpesvirus DNA were subjected to partial DNA sequencing analysis, 100% identical to Acomys herpesvirus SVMS 226,222 from Betaherpesvirinae, which is highly suggestive of rat cytomegalovirus (RCMV). This study has successfully demonstrated the presence of RCMV from Rattus spp. in the Klang Valley. The RCMV potentially crosses species barriers and establishes infection, raising public health concerns. The non-viraemic state of WNV or parvovirus infection, low sample size, and limited niche distribution emphasise the need for the expansion of this study in the future.

Investigating DNA Damage Mechanism Induced by Monosodium Glutamate and Associated DNA Repair Cell Machinery: A Literature Review

Introduction: Monosodium Glutamate (MSG) is a widely used food additive to enhance flavours. Though commonly used, MSG’s accumulation in the body can induce genomic instabilities. These genome instabilities are detectable through various methods such as Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) and comet assay. Additionally, cells can employ DNA repair mechanisms to ameliorate this damage. The objective of this review paper is to investigate the role of prolonged MSG ingestion in DNA damage, potential downstream diseases, and DNA repair mechanisms that cells undertake to counteract these effects, such as nucleotide excision repair (NER) and base excision repair (BER). Compounds such as vitamin C, green tea extract, and Allium sativum have been shown to ameliorate the health hazards of MSG by inhibiting oxidative stress, reducing apoptosis, and increasing antioxidant availability. Methods: This review focuses on MSG-induced DNA damage mechanisms including gene suppression, chromosomal disruption, and carcinogenic effects. We conducted a comprehensive literature review of 28 peer-reviewed articles published since 2000-present. Results: Studies show that MSG consumption may lead to the formation of Reactive Oxygen Species (ROS) and micronuclei (MN), which are known as biomarkers of carcinogenesis. Furthermore, the genomic instabilities that lead to this effect were identified through the use of RAPD-PCR and comet assays. These instabilities are mainly dealt with by endogenous repair machineries such as NER and BER. Also, researchers have identified many substances which act as preventative measures towards the potential harmful impacts of MSG. Discussion: Diseases, such as cancer and obesity, may be linked to chronic intake of MSG. The efficacy of the mentioned DNA damage detection methods will be discussed. Furthermore, the endogenous mechanisms of NER and BER are outlined in this review. Substances such as vitamin C, green tea extract, and Allium sativum aid to prevent genotoxic effects induced by MSG. Conclusion: Through this research, we hope to bring awareness to the harmful impacts of MSG on genome stability and its role in disease development. We aim to educate the public about the prevalent usage of MSG in the food industry and to inform individuals to take precautions in their food consumption.

Molecular Identification of Meloidogyne javanica occurring in Upper Egypt Based Polymerase Chain Reaction (PCR) Assays

Molecular diagnosis represents one of the important trends in the confirmation of morphological identification to the root-knot nematodes which one of the most global pathogens. In this context, current study aimed to describe Meloidogyne javanica occurring in cucumber plants in Assiut governorate (Upper Egypt) using the random amplified polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) assay. Four mitochondrial and specific-specific sequences characterized amplified regions (SCAR) primers were used. The results showed that the SCAR primers, Fjav/Rjav and Far/Rar were efficiently amplified with 670 bp and 517 bp, respectively. On the contrary, the mitochondrial primers did not show any results. In conclusion, the present study recommended the SCAR primers as effective identified primers for the species of Meloidogyne javanica.

Effects of Epstein-Barr Virus Infection on the Risk and Prognosis of Primary Laryngeal Squamous Cell Carcinoma: A Hospital-Based Case-Control Study in Taiwan.

Simple SummaryEpstein–Barr virus DNA positivity, age ≥ 55 years, cigarette smoking, and high BCL-2, B2M, and CD161 expression were identified as independent risk factors for primary laryngeal squamous cell carcinoma. A high EBER signal and low CD3 expression significantly and independently predicted local recurrence and disease-free survival within five years. The information obtained in this study improves our understanding of viral infections in laryngeal cancer, and may guide future prevention, treatment, and follow-up strategies. Mounting molecular evidence supports Epstein–Barr virus (EBV) involvement in the pathogenesis of laryngeal squamous cell carcinoma (LSCC); however, the epidemiological data are inconsistent. In this retrospective case-control study, we aimed to determine whether EBV infection underlies the risk and prognosis of LSCC. The prevalence of EBV infection, as analyzed using an EBV DNA polymerase chain reaction assay, was significantly higher in 42 Taiwanese patients with newly diagnosed primary LSCC, compared to 39 age- and sex-matched control patients without cancer (48% vs. 19%). Furthermore, most of the EBER signals detected using in situ hybridization were localized to the nuclei of tumor-infiltrating lymphocytes. In multivariate analysis, EBV DNA positivity, age ≥ 55 years, cigarette smoking, and high BCL-2, B2M, and CD161 expression (assessed using immunohistochemistry) were identified as independent risk factors for LSCC. Furthermore, five-year local recurrence and disease-free survival rates were 34% and 58%, respectively, with a high EBER signal and low CD3 expression independently predicting five-year local recurrence and disease-free survival. Our comprehensive profiling data accurately identified patients at risk for LSCC development, local recurrence, or disease-free survival. The information obtained in this study improves our understanding of EBV infection in LSCC, and may guide precision medicine for patients with LSCC.

Unexpected Cytomegalovirus (CMV) Replication Kinetics in CMV Donor-Seropositive, Recipient-Seronegative Liver Transplant Recipients Receiving Preemptive Antiviral Therapy.

Detailed cytomegalovirus (CMV) kinetics in donor CMV-seropositive, recipient CMV-seronegative (D+/R-) transplant recipients receiving preemptive therapy (PET) have not been fully defined. The study population consisted of the PET arm of a randomized CMV prevention trial in D+/R- liver transplant recipients. CMV DNA polymerase chain reaction (PCR) assays were performed weekly for 100 days using a sensitive assay. Viral load and clinical parameters were compared for patients with or without high-level increase (defined as higher than the group median log10 increase in viral load from baseline after PET initiation). Among 79 patients, 93.6% (74/79) developed an increase from baseline viral loads of median 120 IU/mL to 3350 IU/mL; 25.7% (19/74) of the patients had peak levels >10 000 IU/mL. None of the patients with rise in viral load underwent testing for CMV resistance, and viremia resolved with PET with valganciclovir. Patients with high-level increase in viral load had a significantly lower rate of recurrent viremia than those without such increase (16/40 [40%] vs 28/39 [71.8%], respectively; P = .004). A majority of D+/R- recipients had a marked increase in viral load after initiation of PET before resolution of viremia. This phenomenon is associated with lower rates of subsequent recurrent viremia and does not necessarily imply antiviral resistance.

Viral Reservoir in Early-Treated Human Immunodeficiency Virus-Infected Children and Markers for Sustained Viral Suppression.

The impact of very early infant treatment on human immunodeficiency virus (HIV) reservoir, and markers for treatment success, require study. The Early Infant Treatment Study (EIT) enrolled 40 children living with HIV started on antiretroviral treatment (ART) at <7 days of age, with 23 who had started treatment between 30-365 days to serve as controls. Quantitative HIV DNA was evaluated every 1-3 months in peripheral blood mononuclear cells. 84-week repeat qualitative whole blood DNA polymerase chain reaction and dual enzyme immunosorbent assay were performed. Median quantitative cell-associated DNA after at least 84 weeks was significantly lower among the first 27 EIT children tested than among 10 controls (40.8 vs 981.4 copies/million cells; P < .001) and correlated with pre-ART DNA. Median DNA after 84 weeks did not differ significantly by negative or positive serostatus at 84 weeks (P = .94), and appeared unaffected by periods of unsuppressed plasma RNA from 24-84 weeks (P = .70). However, negative 84-week serostatus was 67% predictive for sustained RNA suppression, and positive serostatus was 100% predictive for viremia. Loss of qualitative DNA positivity at 84 weeks was 73% predictive for sustained suppression, and persistent positivity was 77% predictive for viremia. Lower viral reservoir was associated with starting ART at <1 week. Negative serostatus and qualitative DNA were useful markers of sustained viral suppression from 24-84 weeks.

Direct Amplification of Whole Blood and Amniotic Fluid Specimens for Prenatal and Postnatal Diagnosis of Hb E-β 0-Thalassemia Diseases.

Prenatal and postnatal diagnosis of hemoglobin E-β 0-thalassemia can be made using polymerase chain reaction (PCR) analysis mostly on purified DNA. We have establihed a direct amplification method without DNA extraction on whole blood (WB) and amniotic fluid (AF) specimens to diagnose the disease. Three reactions of WB PCR assays and 7 reactions of AF PCR tests were developed for postnatal and prenatal diagnosis, respectively. Assays were validated against routine tests in a blinded trial. The results showed 100% concordance with routine DNA PCR assays. Among 309 β-thalassemia carriers, 191 patients (61.8%) carried common β-thalassemia mutations. Among 448 AF specimens, 116 (25.9%) fetuses were found to be affected, 247 (55.1%) fetuses were carriers, and 85 (19%) fetuses were unaffected. We found that WB and AF PCR assays are simple, rapid, and reliable. The developed techniques could be applicable in routine settings.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

Insecticidal and Genotoxic effects of some indigenous plant extracts in Culex quinquefasciatus Say Mosquitoes

Five different weed plants viz. Convulvulus arvensis, Chenopodium murale, Tribulus terrestris, Trianthema portulacastrum, and Achyranthes aspera were investigated for their entomocidal and genotoxic effects against Culex quinquefasciatus mosquitoes. High mortality was observed at 72 hours in a dose dependent manner. Among all the tested plants, A. aspera was found highly significant which showed 100% mortality at 250 ppm after 72 hours with LC50 of 87.46, 39.08 and 9.22 ppm at 24, 48, respectively. In combination with Bacillus thuringiensis israelensis (Bti); A. aspera also caused 100% mortality at 250 ppm concentration after 72 hours (LC50 8.29 ppm). Phytochemical analysis of all the tested weed plants showed the presence of flavonoids, saponins, tannins, steroids, cardiac glycosides, alkaloids, anthrequinones and terpenoids. Random Amplification of Polymorphic DNA-Polymerase chain reaction (RAPD-PCR) and comet assay were performed to assess the genotoxic effect of A. aspera but no change in DNA profile was observed. Furthermore, FTIR showed the presence of phenolic compounds in A. aspera extract. It is suggested that certain phenolic compounds such as flavonoids modulate the enzymatic activity and, hence, cause the death of larvae of Cx. quinquefasciatus. Altogether, current study would serve as an initial step towards replacement of synthetic insecticides to plant-microbe based biopesticide against Culex mosquitoes in future.

Application of multiplex PCR assay to study early multiple herpesviruses infection during HSCT

目的采用多重PCR方法分析造血干细胞移植（HSCT）患者多种疱疹病毒的感染状况，并探讨HSCT患者多种疱疹病毒感染与临床特征的相关性及其对移植并发症和预后的影响。方法以苏州大学附属第一医院血液科2017年2月至2017年8月行HSCT的90例患者为研究对象，收集预处理至移植后90 d内不同时间点的外周血标本共734份，Lab-Aid824核酸提取Mini试剂抽提DNA，应用多重PCR方法同时扩增8种人类疱疹病毒，分析多种疱疹病毒感染发生率及其与临床特征的相关性及对移植后并发症和预后的影响。结果至随访终点，中位随访时间为192（35~308）d。移植前疱疹病毒感染发生率为35.6%（32/90），其中1种疱疹病毒感染发生率为12.2%（11/90），多种病毒感染的发生率为23.3%（21/90）。移植后疱疹病毒感染发生率为77.8%（70/90），其中1种疱疹病毒感染发生率为20.0%（18/90），多种疱疹病毒感染的发生率为57.8%（52/90）。在多种疱疹病毒感染的患者中，2种病毒感染30例（57.7%），3种疱疹病毒感染18例（34.6%），不同时间点样本检测的4种疱疹病毒感染4例（7.7%）。移植后多种疱疹病毒感染中，HHV-6和HHV-7感染存在相关性（OR=13.880，Q=0.026），EBV和HHV-7感染也存在相关性（OR=0.093，Q=0.044）。25例患者移植后出现疱疹病毒感染相关临床表现，主要为出血性膀胱炎、间质性肺炎、肠炎、病毒性脑炎和不明原因发热。移植前HHV-1感染与年龄、HHV-2感染与发病时间、CMV感染与原发病为淋巴瘤具有一定的相关性。移植后EBV感染与HLA不全相合、供受者ABO血型不一致及Ⅱ~Ⅳ度急性GVHD呈正相关；多种疱疹病毒感染与HLA不全相合、非血缘供者及Ⅱ~Ⅳ度急性GVHD呈正相关。结论HSCT前后存在多种疱疹病毒感染，HLA不全相合、非血缘供者和Ⅱ~Ⅳ度急性GVHD与移植后多种疱疹病毒感染存在一定的相关性。

Analyses of the genetic diversity and population structures of Histoplasma capsulatum clinical isolates from Mexico, Guatemala, Colombia and Argentina, using a randomly amplified polymorphic DNA-PCR assay.

We studied the genetic diversity and the population structure of human isolates of Histoplasma capsulatum, the causative agent of histoplasmosis, using a randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) assay to identify associations with the geographic distribution of isolates from Mexico, Guatemala, Colombia and Argentina. The RAPD-PCR pattern analyses revealed the genetic diversity by estimating the percentage of polymorphic loci, effective number of alleles, Shannon's index and heterozygosity. Population structure was identified by the index of association (IA) test. Thirty-seven isolates were studied and clustered into three groups by the unweighted pair-group method with arithmetic mean (UPGMA). Group I contained five subgroups based on geographic origin. The consistency of the UPGMA dendrogram was estimated by the cophenetic correlation coefficient (CCCr = 0.94, P = 0.001). Isolates from Mexico and Colombia presented higher genetic diversity than isolates from Argentina. Isolates from Guatemala grouped together with the reference strains from the United States of America and Panama. The IA values suggest the presence of a clonal population structure in the Argentinian H. capsulatum isolates and also validate the presence of recombining populations in the Colombian and Mexican isolates. These data contribute to the knowledge on the molecular epidemiology of histoplasmosis in Latin America.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

Bionomics of phlebotomine sand flies species (Diptera: Psychodidae) and their natural infection with Leishmania and Crithidia in Fars province, southern Iran.

Phlebotominae sand flies are involved in human diseases, such as leishmaniasis, and cause a considerable number of deaths every year. Besides, some of them have been identified as allergen sources or the potential mechanical vectors related to nosocomial infections. The present study aimed to assess the monthly activity, fauna, and detection of protozoan agents in phlebotomine sand flies using polymerase chain reaction (PCR) in re-emerging zoonotic cutaneous leishmaniasis foci of Shiraz and Kharameh in Fars province, southern Iran during 2016-2017. To determine the monthly activity, sand flies were caught from indoors and outdoors of both studied areas. Afterward, all female phlebotomine sand flies were processed for DNA extraction and PCR assays for Leishmania and Crithidia detections. During the study, 6975 sand flies of 16 species (eight Phlebotomus and eight Sergentomyia species) were caught in both foci. Sand flies' monthly activities started in early April and terminated in late November and October. Additionally, two active peaks of sand flies were observed in both foci; first in June and second in August to September. Phlebotomus papatasi (47.1%) was the most dominant species in out/indoors of both Shiraz (31.1%) and Kharameh (16.0%). It was also the only species which was found infected with Leishmania major, indeed, 2.68% and 2.53% of P. papatasi were infected to L. major in Kharameh and Shiraz, respectively. However, none of the female sand flies was positive for Crithidia spp. Despite various control strategies, especially against Leishmania, considerable cases of leishmaniasis are recorded from Iran every year. Phlebotomine plays the main role in transmission of Leishmania in these foci. Therefore, further studies are needed to determine the role of different phlebotomine species in epidemiological aspects of leishmaniasis.

The importance of morphological identification of African anopheline mosquitoes (Diptera: Culicidae) for malaria control programmes

BackgroundThe correct identification of disease vectors is the first step towards implementing an effective control programme. Traditionally, for malaria control, this was based on the morphological differences observed in the adults and larvae between different mosquito species. However, the discovery of species complexes meant that genetic tools were needed to separate the sibling species and today there are standard molecular techniques that are used to identify the two major malaria vector groups of mosquitoes. On the assumption that species-diagnostic DNA polymerase chain reaction (PCR) assays are highly species-specific, experiments were conducted to investigate what would happen if non-vector species were randomly included in the molecular assays.MethodsMorphological keys for the Afrotropical Anophelinae were used to provide the a priori identifications. All mosquito specimens were then subjected to the standard PCR assays for members of the Anopheles gambiae complex and Anopheles funestus group.ResultsOne hundred and fifty mosquitoes belonging to 11 morphological species were processed. Three species (Anopheles pretoriensis, Anopheles rufipes and Anopheles rhodesiensis) amplified members of the An. funestus group and four species (An. pretoriensis, An. rufipes, Anopheles listeri and Anopheles squamosus) amplified members of the An. gambiae complex.ConclusionsMorphological identification of mosquitoes prior to PCR assays not only saves time and money in the laboratory, but also ensures that data received by malaria vector control programmes are useful for targeting the major vectors.

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

AbstractAn efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape.

Keratinizing odontogenic cysts with a spectrum of verrucoid morphology: investigation of a potential role of human papillomavirus

The role of human papillomavirus (HPV) in keratinizing odontogenic cysts (OC) has only rarely been studied. We describe the clinicopathologic findings in a series of OCs that had unusual keratinization patterns and were investigated for a possible HPV etiology. Tissue samples from 29 patients with keratinizing OCs were studied for light microscopic features suggestive of HPV infection and by an HPV DNA polymerase chain reaction assay. The mean age at presentation was 31.1years; 79.3% of the OCs occurred in the mandible and 46.4% were associated with an impacted tooth. The phenotypic characteristics koilocytes, hypergranulosis, and a verrucous pattern of the cyst-lining epithelium were observed in 69%, 62.1%, and 17.2% of cases, respectively. These histomorphologic features did not, however, correlate with HPV infection. HPV does not appear to play a role in keratinizing OCs and is not responsible for the wart-like histomorphologic features that may be seen in these lesions.

Clinical Significance of MYD88 Mutation in Patients with Diffuse Large Cell Lymphoma

Mutation of the MYD88 has recently been identified in activated B cell like diffuse large B cell lymphoma (DLBCL) and enhanced cell proliferation systems such as JAK-STAT and NF-kB signaling pathways. However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive and an automatic method utilizing guanine-quenching probes (QP) to detect mutation and investigated the relationship between MYD88 L265P mutation and clinical significance. We amplify a DNA fragment including the mutation to intend for by PCR and associate it with Q-probe with complementary sequence, using the temperature that Q-probe dissociates varying according to a conformity degree of the complementarity sequence. We judge it by detecting the fluorescence to be provided by dissociation. Results were obtained from 1ul of DNA solution(10ng) within 90 min by the method. Detected mutations were identical between QP method and allele-specific PCR (AS-PCR).Eighty-nine patients with a diagnosis of de novo DLBCL made between 1999 and 2014, and treated with CHOP or R-CHOP therapy. We retrospectively analyzed the outcome of 89 patients (age range; 21-88 and 59% were female). The median follow-up time was 4.4 y. Survival analyses were performed using the Kaplan-Meier method. None of the patients had a known history of human immunodeficiency virus infection. MYD88 L265P mutation was both assessed by Q-probe system that can detect low levels of mutant DNA and allele-specific TaqMan polymerase chain reaction assay. We performed the direct sequence method using 3130 Applied Biosystem Genetic Analyzer as antithesis. The cell-of-origin was determined based on immunohistochemical (IHC) stains for CD10, BCL-6 and MUM-1 by Hans' algorithm.MYD88 L265Pmutation was detected in 25.8% (23/89) in various tissues of DLBCL. MYD88 mutations occurred more frequently in males (P<0.05), cases without B symptoms (P<0.05). MYD88 mutation was infrequent in DLBCL arising in lymph nodes (10.6%), but more frequently found in extranodal sites such as testes (83%, 5/6), nasal (75%,9/12), central nervous system (50%,2/4), and leg (100%,1/1). In agreement with recent studies, we found no mutated cases among gastric cases. As somatic mutations in MYD88 was reported to be the most frequent alterations found in non-GCB type, we further analyzed GCB or non-GCB type by IHC. MYD88 mutations were predominantly observed in the non-GCB type (74%, 17/23), compared with 26%, 6/23 in GCB type. Overall survival (OS) for 3 years were 84.2% and 70.2% in patients with wild-type MYD88 and in MYD88 mutation group (P=0.366), respectively. Progression-free survival (PFS) for 3 years, 76.9% and 64.3% in patients with wild type and in mutated group (P=0.156), respectively. However, all four cases with CNS relapse had this mutation, 2 originated from testis, and remained 2 from lymph nodes. Our results confirm the remarkable site-specific occurrence of MYD88 mutation. In addition, Q-probe system for detection of MYD88 mutation was very useful because of its sensitivity and in the case who obtained only a small amount of biopsy specimen. MYD88 L265Ppromotes survival of malignant lymphoid cells through several mechanisms. Further large scale study should be necessary for more understanding of biological and clinical significance of DLBCL patients with MYD88 mutation. DisclosuresNo relevant conflicts of interest to declare.