Simultaneous Detection of Major Pome Fruit Viruses and a Viroid

A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of ~198, ~330, ~370, ~547 and ~645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.     

Occurrence and Diversity of Pome Fruit Viruses in Apple and Pear Orchards in Latvia

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes.     

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

Detection of Prunus necrotic ring spot virus in plum,cherry and almond by serological and molecular techniques from India

Stone fruits are cultivated in the temperate and sub-temperate regions of India. During surveys in stone fruit growing areas, viral symptoms were observed in almond, cherry and plum. These samples were brought to the laboratory for further detection at serological and molecular levels to check the presence of virus. In the present study, incidence of PNRSV is reported on plum (Prunus domestica), almond (Prunus dulcis) and cherry (Prunus avium) using serological and molecular techniques. Coat protein gene of PNRSV was amplified from almond, cherry and plum. This is the first molecular evidence of PNRSV on these stone fruits reported from India.     

Influence of in vitro factors on titre and elimination of model fruit tree viruses

The titre of apple chlorotic leaf spot virus (ACLSV) was higher in microplants of Malus domestica cv. Jonagold than in 2-year-old grafted scions. Cytokinin concentration in the medium increased the titre of prunus necrotic ringspot virus (PNRSV) in the apex of microplants of Prunus insititia cv. Kozlienka but did not affect the titre of ACLSV in M .domestica.Virus titre of ACLSVwas higher in the haulms of autotrophically-grown compared with heterotrophically-grown microplants whereas as for PNRSV the results were the reverse. For both viruses, however, titre of the virus in the roots of autotrophically-grown plants was significantly higher than in haulm tissue from heterotrophic cultures. Ribavirin incorporation resulted in elimination of both viruses. Negative ELISA results were confirmed independently by PCR. The efficacy of Ribavirin in elimination of ACLSV was increased by increasing the concentration of cytokinin in the medium in parallel with decreasing the concentration of Ribavirin. These results are discussed in the context of the reliability of in vitro virus testing.     

Incidence of Prunus necrotic ring spot virus on Malus domestica in India

In surveys of apple (Malus domestica) orchards in various parts of Himachal Pradesh, samples from trees showing necrotic symptoms on the leaves were collected and tested for detection of Prunus necrotic ringspot virus (PNRSV) initially by ELISA followed by RT‐PCR using coat protein gene primers. Positive results were obtained in samples from Kullu and Kalpa regions. The virus gene sequences showed 88–97% similarity to corresponding sequences of other PNRSV isolates deposited in the GenBank database using ncbi.nih.nlm.gov. Although the similarity was high, there were some distinct differences with the Spanish isolate. This is the first report of PNRSV in apple from India.     

Molecular pathology in the diagnosis and treatment of non-Hodgkin's lymphomas

The non-Hodgkin's lymphomas encompass a wide spectrum of hematologic neoplasms that exhibit different clinical and biological features. Lymphomas classically have been initially assessed based on their cytologic and histologic features. Morphology alone is often inadequate as similar appearing neoplasms may be immunophenotypically and molecularly heterogeneous. Molecular diagnostic methods can provide an additional level of testing that not only helps refine diagnoses but can provide prognostic information. New methods are being refined that may provide information to establish precise diagnostic profiles, provide targets for therapy and provide more sensitive methods for monitoring the success of treatment. Molecular methods will be increasingly utilized and eventually required as the accepted method of diagnosis and for monitoring the disease. Understanding of the molecular abnormality and the pathogenesis of the neoplasm hopefully will lead to therapeutic intervention aimed at the specific molecular defect or its product. The molecular pathology of the non-Hodgkin's lymphomas is discussed.     

Molecular diagnosis in oncology

Recent advances of molecular genetics have exerted a noticeable effect on some areas of clinical medicine. A comprehensive understanding of the nature of hereditary tumors is frequently heralded as the most substantial practical achievement of molecular oncology. Proper diagnostic algorithms have already been developed for the vast majority of known familial cancer syndromes. Interestingly, an unexpectedly strong founder effect has been documented at least for some hereditary cancers occurring in Russia, which significantly simplifies the detection of the corresponding disease-associated gene variants. The number of tests aimed at customizing cancer treatment continues to grow every year. The EGFR mutation test is probably the most impressive one, as it predicts the lung cancer response or nonresponse to gefitinib or erlotinib with a really unprecedented accuracy. Approaches helping to determine the individual efficacy and safety profiles for fluoropyrimidines, platinum compounds, irinotecan, etc. are currently under development. Methods detecting residual amounts of disseminated cancer cells represent another popular avenue of research. These technologies are expected to improve the quality of prediction of local and distant metastases, facilitate monitoring of the minimal residual disease, and, in the long-term perspective, provide a tool for early cancer diagnosis. It should be emphasized that molecular detection of disseminated tumor cells is currently used mainly in research settings and is not yet incorporated into routine clinical practice.     

病毒分子生物学诊断方法的应用进展

作为病毒诊断实验室必备的一种手段 ,PCR相关的病毒分子生物学诊断技术准确、快速、敏感、简便 ,对指导临床治疗十分重要。本文对近年来 PCR技术方法学研究与应用研究及生物传感器诊断病毒等方面的一些进展进行了综述     

Molecular diagnosis of Helicobacter pylori antibiotic resistance in the Taif region,Saudi Arabia

Success in eradication of Helicobacter pylori is declining globally because H. pylori has developed resistance against most of the antibiotics proposed for eradication regimens, mainly through point mutations. The present study included 200 patients with dyspepsia attending Taif Hospital. Gastric biopsies were obtained during gastroscopy and subjected to rapid urease testing. Molecular methods were used to confirm diagnoses of H. pylori infection and to identify resistance gene variants of four antibiotics; namely, clarithromycin, metronidazole, fluoroquinolones and tetracycline (23S rRNA, gyrA, rdxA and 16S rRNA respectively). Of all investigated patients, Molecular diagnoses were made in 143 of all investigated patients; thus, the prevalence was .5%. The overall rate of resistance to clarithromycin among the H. pylori‐positive patients was high (39.9%) and the rate of resistance significantly greater (48.2%) among the secondary resistance group, secondary resistance being defined as resistance as a result of previous exposure to the relevant antibiotic. The rate of resistance to fluoroquinolones was considered moderate; the difference in rate of resistance between the primary and secondary resistance groups (8.4% and 9.5%, respectively) was not significant Also, there was a low prevalence of both primary and the secondary tetracycline resistance in the study cohort. In contrast, the prevalence of metronidazole resistance was considered high with no significant difference between the two resistance groups. H. pylori showed an increased prevalence of resistance to all four of the commonly used therapeutic agents. Thus, eradication therapy should be based on the regional results of susceptibility testing. Moreover, treatment tailored according to individually determined H. pylori susceptibility may be a reasonable future goal.     

Biological and Molecular Characterization of Three Isolates of Tomato aspermy virus Infecting Chrysanthemums in India

Severe mosaic, chlorotic ringspots and flower deformation were observed during the winter of November 2006–February 2007 on chrysanthemums ( Chrysanthemum morifolium ) at three locations in India: Lucknow (UP), Dhanbad (MP) and Kolkata (WB). Tomato aspermy virus (TAV) was detected in affected plants by ELISA and by RT-PCR using TAV specific primers. These TAV isolates were mechanically transmitted to test plant species and also by aphids ( Aphis gossypii ) to Lycopersicon esculentum . The complete RNA 3 of each TAV isolate was cloned and sequenced and determined to be 2386 nucleotides (nt) long, and to encode two open reading frames (ORFs): the movement protein (MP) of 741 nt and the coat protein (CP) of 657 nt translating in to 246 and 218 amino acid (aa), respectively. When RNA 3 sequences of the Indian isolates were multiple aligned with seven other strains of TAV occurring worldwide, Indian isolates shared 98–99% identities among themselves and with the KC, V, P, B, I and C strains of TAV. In phylogenetic analysis, the Lucknow and Kolkata isolates of TAV clustered together and showed a close relationship with the KC-TAV strain from South Korea, whereas the Dhanbad isolate formed an independent cluster and showed closeness with the V-TAV strains from Spain and Australia. Recombination events were also observed in the CP region of the Dhanbad isolate, supporting its diverse behaviour. This is the first report of the complete RNA 3 sequence of these three Indian TAV isolates.     

Nucleotide sequence and secondary structure of apple scar skin viroid.

The complete nucleotide sequence of apple scar skin viroid(ASSV) has been established, and a probable secondary structure is proposed. A single-stranded circular ASSV RNA consists of 330 nucleotides and can assume the rodlike conformation with extensive base-pairing characteristic of all the known viroids. ASSV shows low sequence homologies with other viroids and lacks the central conserved region. These indicate that ASSV should be allocated to a separate viroid group. However, homologous sequences with potato spindle tuber viroid(PSTV) in ASSV occur in limited and scattered regions of both viroids. These homologous regions fall within the particular domains in the viroid domain model which has been previously proposed by Keese and Symons(Proc. Natl. Acad. Sci. USA. 82, 4582-4586, 1985).     

基于CRISPR/Cas的分子诊断传感器在非核酸分子诊断中的应用

CRISPR/Cas不仅是一种重要的基因编辑工具,而且还是一种有效的分子诊断工具。目前基于CRISPR/Cas建立了一系列的分子诊断传感器系统,广泛应用于核酸、非核酸等检测过程中。与应用较广泛的核酸分子诊断传感器系统相比,基于CRISPR/Cas的非核酸检测系统目前尚未见系统性综述,因此本文围绕基于CRISPR/Cas12和CRISPR/Cas13建立的两大类非核酸分子传感器诊断系统的基本特征、工作流程及其检测原理等进行了全面综述,期望能为CRISPR/Cas分子诊断系统在体外诊断中的应用提供依据。     

Biological and molecular characterisation of Prunus necrotic ringspot virus isolates and possible approaches to their phylogenetic typing

Seven isolates of Prunus necrotic ringspot virus (PNRSV) originating from Slovakia were subjected to biological tests under glasshouse conditions. Mainly mild symptoms were observed on chip‐budded test cherry rootstocks. The complete sequence for the capsid protein (CP) gene of four isolates was determined. All sequences were 675 nucleotides long and clustered in the largest of four groups delineated by phylogenetic analyses of all so far known PNRSV CP sequences. A set of restriction endonucleases was suggested to differentiate four isolate clusters by restriction enzyme digestion of CP sequences.     

珊瑚病原微生物鉴定及其分子诊断技术进展

珊瑚礁生态系统是热带海洋最突出、最具代表性的生态系统,具有极高的生态价值和经济价值,然而由珊瑚疾病引起的珊瑚礁退化已经成为珊瑚礁生态系统的主要威胁之一。许多微生物(主要包括细菌、真菌、病毒)被认为与珊瑚疾病发生密切相关,确定珊瑚疾病的病原并建立其快速诊断的方法是开展珊瑚疾病流行病学调查和制定防控措施的必由之路。本文主要综述珊瑚疾病的病原微生物及其分子诊断技术的研究进展。     

Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5–10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.