Reverse Transcription-polymerase Chain Reaction Protocol Research Articles

A Multiplex Nanopore Sequencing Approach for the Detection of Multiple Arboviral Species.

The emergence and continued geographic expansion of arboviruses and the growing number of infected people have highlighted the need to develop and improve multiplex methods for rapid and specific detection of pathogens. Sequencing technologies are promising tools that can help in the laboratory diagnosis of conditions that share common symptoms, such as pathologies caused by emerging arboviruses. In this study, we integrated nanopore sequencing and the advantages of reverse transcription polymerase chain reaction (RT-PCR) to develop a multiplex RT-PCR protocol for the detection of Chikungunya virus (CHIKV) and several orthoflaviviruses (such as dengue (Orthoflavivirus dengue), Zika (Orthoflavivirus zikaense), yellow fever (Orthoflavivirus flavi), and West Nile (Orthoflavivirus nilense) viruses) in a single reaction, which provides data for sequence-based differentiation of arbovirus lineages.

Assessing severe acute respiratory syndrome coronavirus 2 infectivity by reverse-transcription polymerase chain reaction: A systematic review and meta-analysis.

The cornerstone of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) detection is reverse‐transcription polymerase chain reaction (RT‐PCR) of viral RNA. As a surrogate assay SARS‐CoV‐2 RNA detection does not necessarily imply infectivity. Only virus isolation in permissive cell culture systems can indicate infectivity. Here, we review the evidence on RT‐PCR performance in detecting infectious SARS‐CoV‐2. We searched for any studies that used RT‐PCR and cell culture to determine infectious SARS‐CoV‐2 in respiratory samples. We assessed (i) diagnostic accuracy of RT‐PCR compared to cell culture as reference test, (ii) performed meta‐analysis of positive predictive values (PPV) and (iii) determined the virus isolation probabilities depending on cycle threshold (Ct) or log10 genome copies/ml using logistic regression. We included 55 studies. There is substantial statistical and clinical heterogeneity. Seven studies were included for diagnostic accuracy. Sensitivity ranged from 90% to 99% and specificity from 29% to 92%. In meta‐analysis, the PPVs varied across subgroups with different sampling times after symptom onset, with 1% (95% confidence interval [CI], 0%–7%) in sampling beyond 10 days and 27% (CI, 19%–36%) to 46% (CI, 33%–60%) in subgroups that also included earlier samples. Estimates of virus isolation probability varied between 6% (CI, 0%–100%) and 50% (CI, 0%–100%) at a Ct value of 30 and between 0% (CI, 0%–22%) and 63% (CI, 0%–100%) at 5 log10 genome copies/ml. Evidence on RT‐PCR performance in detecting infectious SARS‐CoV‐2 in respiratory samples was limited. Major limitations were heterogeneity and poor reporting. RT‐PCR and cell culture protocols need further standardisation.

Point-of-Care Diagnostic Tools for Surveillance of SARS-CoV-2 Infections.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a recently emerged and highly contagious virus that causes coronavirus disease 2019 (COVID-19). As of August 24, 2021, there were more than 212 million confirmed COVID-19 cases and nearly 4.4 million deaths reported globally. Early diagnosis and isolation of infected individuals remains one of the most effective public health interventions to control SARS-CoV-2 spread and for effective clinical management of COVID-19 cases. Currently, SARS-CoV-2 infection is diagnosed presumptively based on clinical symptoms and confirmed by detecting the viral RNA in respiratory samples using reverse transcription polymerase chain reaction (RT-PCR). Standard RT-PCR protocols are time consuming, expensive, and technically demanding, which makes them a poor choice for large scale and point-of-care screening in resource-poor settings. Recently developed isothermal nucleic acid amplification tests (iNAAT), antigen and/or serological tests are cost-effective to scale COVID-19 testing at the point-of-care (PoC) and for surveillance activities. This review discusses the development of rapid PoC molecular tools for the detection and surveillance of SARS-CoV-2 infections.

Low clinical performance of the Isopollo COVID-19 detection kit (M Monitor, South Korea) for RT-LAMP SARS-CoV-2 diagnosis: A call for action against low quality products for developing countries

BackgroundMultiple molecular kits are available for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide, with many lacking proper clinical evaluation due to the emergency caused by the coronavirus disease 2019 (COVID-19) pandemic, particularly in developing countries. MethodsThis study was conducted to evaluate the clinical performance of the Isopollo COVID-19 detection kit (M Monitor, South Korea) for reverse transcription loop-mediated isothermal amplification (RT-LAMP) SARS-CoV-2 diagnosis, using the SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) protocol as the gold standard. ResultsA total of 220 clinical samples were included in the study; 168 samples were SARS-CoV-2-positive and 52 samples were SARS-CoV-2-negative according to the SARS-CoV-2 RT-PCR protocol. For the Isopollo COVID-19 detection kit, only 104 out of 168 samples were SARS-CoV-2-positive. This result shows a low clinical performance, with sensitivity of 61.9% for the evaluated RT-LAMP assay. ConclusionsProper clinical performance evaluation studies by regulatory agencies in developing countries such as Ecuador should be mandatory prior to clinical use authorization of SARS-CoV-2 diagnosis kits, particularly when those kits lack either US Food and Drug Administration or country of origin clinical use authorization.

Transmission of SARS-COV-2 Infections in Households - Tennessee and Wisconsin, April-September 2020.

Improved understanding of transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), within households could aid control measures. However, few studies have systematically characterized the transmission of SARS-CoV-2 in U.S. households (1). Previously reported transmission rates vary widely, and data on transmission rates from children are limited. To assess household transmission, a case-ascertained study was conducted in Nashville, Tennessee, and Marshfield, Wisconsin, commencing in April 2020. In this study, index patients were defined as the first household members with COVID-19-compatible symptoms who received a positive SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) test result, and who lived with at least one other household member. After enrollment, index patients and household members were trained remotely by study staff members to complete symptom diaries and obtain self-collected specimens, nasal swabs only or nasal swabs and saliva samples, daily for 14 days. For this analysis, specimens from the first 7 days were tested for SARS-CoV-2 using CDC RT-PCR protocols.† A total of 191 enrolled household contacts of 101 index patients reported having no symptoms on the day of the associated index patient's illness onset, and among these 191 contacts, 102 had SARS-CoV-2 detected in either nasal or saliva specimens during follow-up, for a secondary infection rate of 53% (95% confidence interval [CI]=46%-60%). Among fourteen households in which the index patient was aged <18 years, the secondary infection rate from index patients aged <12 years was 53% (95% CI=31%-74%) and from index patients aged 12-17 years was 38% (95% CI=23%-56%). Approximately 75% of secondary infections were identified within 5 days of the index patient's illness onset, and substantial transmission occurred whether the index patient was an adult or a child. Because household transmission of SARS-CoV-2 is common and can occur rapidly after the index patient's illness onset, persons should self-isolate immediately at the onset of COVID-like symptoms, at the time of testing as a result of a high risk exposure, or at the time of a positive test result,whichever comes first. Concurrent to isolation, all members of the household should wear a mask when in shared spaces in the household.§.

Transmission of SARS-COV-2 Infections in Households — Tennessee and Wisconsin, April–September 2020

Improved understanding of transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), within households could aid control measures. However, few studies have systematically characterized the transmission of SARS-CoV-2 in U.S. households (1). Previously reported transmission rates vary widely, and data on transmission rates from children are limited. To assess household transmission, a case-ascertained study was conducted in Nashville, Tennessee, and Marshfield, Wisconsin, commencing in April 2020. In this study, index patients were defined as the first household members with COVID-19-compatible symptoms who received a positive SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) test result, and who lived with at least one other household member. After enrollment, index patients and household members were trained remotely by study staff members to complete symptom diaries and obtain self-collected specimens, nasal swabs only or nasal swabs and saliva samples, daily for 14 days. For this analysis, specimens from the first 7 days were tested for SARS-CoV-2 using CDC RT-PCR protocols.† A total of 191 enrolled household contacts of 101 index patients reported having no symptoms on the day of the associated index patient's illness onset, and among these 191 contacts, 102 had SARS-CoV-2 detected in either nasal or saliva specimens during follow-up, for a secondary infection rate of 53% (95% confidence interval [CI] = 46%-60%). Among fourteen households in which the index patient was aged <18 years, the secondary infection rate from index patients aged <12 years was 53% (95% CI = 31%-74%) and from index patients aged 12-17 years was 38% (95% CI = 23%-56%). Approximately 75% of secondary infections were identified within 5 days of the index patient's illness onset, and substantial transmission occurred whether the index patient was an adult or a child. Because household transmission of SARS-CoV-2 is common and can occur rapidly after the index patient's illness onset, persons should self-isolate immediately at the onset of COVID-like symptoms, at the time of testing as a result of a high risk exposure, or at the time of a positive test result, whichever comes first. Concurrent to isolation, all members of the household should wear a mask when in shared spaces in the household.§.

Preliminary Identification of Human Leukocyte Antigen (HLA) CLASS II DRB1 Allelic Variants in Selected Filipino Cancer Patient Samples

Objective. The Human Leukocyte Antigen (HLA) Class II is the major histocompatibility complex surface glycoproteins of humans responsible for presenting exogenous antigenic peptides which help direct specificity of immune response. In immune-cell therapy, the HLA allelic variants are of particular importance as they determine the successful activation of target cells that results to a desired therapeutic response. However, HLA Class II exhibits high polymorphism and has variable distribution in population, constituting these so-called allelic variants. Specifically, the HLA Class II DRB1 is considered the predominant locus among Filipinos. This research aimed to identify the presence of HLA Class II DRB1 allelic variants in the stem cell samples of ten (10) Filipino cancer patients by reverse transcription polymerase chain reaction (RT-PCR) amplification.Method. This study employed a PCR-based HLA Class II typing to identify the HLA Class II DRB1 allelic variant in Filipino cancer patients. Design of forward and reverse primers for HLA Class II DRB1, optimization of PCR conditions for amplifying HLA Class II DRB1, and identification of HLA Class II DRB1 allelic variants from samples by sequencing and database comparison were conducted.Results. PCR optimization showed that optimum annealing temperature for HLA DRB1 was 58.8°C with 1 mM MgCl2. PCR amplification of HLA DRB1 from ten anonymized cancer patient samples and DNA sequencing revealed that Patients 1, 2, 5, 8, 9, and 10 harbor HLA DRB1 allelic variants, particularly, the HLA DRB1*04:06:01, HLA DRB1*12:01:01, HLA DRB1*0813, HLA DRB1*04:05:01, HLA DRB1*09:01:02, and HLA DRB1*16:02:01, allelic variants, respectively.Conclusion. Using the designed primers and optimized RT-PCR protocol, HLA information derived from six out of ten patient samples can be used for further applications in developing personalized or generic antigenic peptides such as dendritic cell cancer vaccine.

Barley yellow dwarf virus-PAV is the dominant species causing Barley yellow dwarf disease in South Dakota and Minnesota

Barley yellow dwarf (BYD), caused by ten different species of viruses from the Luteoviridae family, is an economically important viral disease of small grains in the US Northern Great Plains. Identification of virus species in the region associated with BYD is critical to deployment of specific effective resistance genes. However, a comprehensive survey of virus species using modern diagnostic tools has not been done before in the US Northern Great Plains. In this study, species composition of viruses associated with BYD in small grains of Minnesota and South Dakota were determined by sampling BYD infected tissue from a large geographical area over three years. Whole genomic RNA was extracted from leaf tissue and reverse transcription polymerase chain reaction (RT-PCR) was conducted to detect infection of BYD and classify into species using primers from a two-step multiplex PCR protocol. Out of 463 samples collected from Minnesota and South Dakota from 2013 to 2015, about 50% (n = 222) of the samples tested positive for BYD. The majority of virus infected samples (n = 146) were identified as the Barley yellow dwarf virus PAV. These results indicate that the RT-PCR protocol used in this study is a viable molecular diagnostic tool for detection of BYD causing viruses in field collected samples. Moreover, the results indicate that Barley yellow dwarf virus PAV is most the dominant species associated with BYD in the US Northern Great Plains. Due to the species-specific nature of resistance to BYD these results can also guide breeding strategies in small grains for BYD management.

First detection and characterization of Psittaciform bornaviruses in naturally infected and diseased birds in Thailand

In Thailand a proventricular dilation disease (PDD)-like syndrome commonly occurs in captive psittacine birds. The etiology, however, has been unknown to date and studies to detect parrot bornaviruses have never been performed in Southeastern Asia. Therefore, 111 psittacines (22 different species) including birds with suspected PDD based on clinical examination results (n = 65), cage mates of PDD suspected parrots without any clinical signs (n = 39) and dead birds with previous clinic suspicious for PDD (n = 7) were tested for bornaviruses using various reverse transcription polymerase chain reaction (RT-PCR) and realtime RT-PCR protocols, an enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and genome sequencing. Bornaviral infections, indicated by the presence of RNA or antibody positive reactions were detected in 60 birds (54.1%) belonging to 15 psittaciform species and originating from 41 owners. Occurrence of Psittaciform 1 orthobornavirus was confirmed by sequencing of PCR products in 24 of these birds. Parrot bornavirus (PaBV)-5, belonging to the species Psittaciform 2 orthobornavirus and found only in single birds in the United States of America, Japan and Hungary until now, was identified in a macaw. Full genome sequencing revealed features shared with other strains of this virus. PaBV-4 was the prevalent virus type and the viruses grouped in two of the five genetic PaBV-4 subclusters known so far while PaBV-2 was found in a single patient. Forty-five psittacines of the group of PDD-suspected birds (69.2%), 4 dead birds and 11 clinically healthy cage mates were positive in at least one test the latter suggesting inefficient horizontal transmission in natural infections. Lymphoplasmacytic infiltrations (non-purulent inflammation, ganglioneuritis) and bornavirus antigen were detected in diverse tissues confirming PDD as the disease involved. These results may have a major impact on conservation projects including the five near-threatened parrot species living in the wild in Thailand.

Diagnostic Reliability of Different RT-PCR Protocols for the Detection of Bluetongue Virus Serotype 14 (BTV-14).

IntroductionThe reverse transcription polymerase chain reaction (RT-PCR) is one of the most extensively used methods for identification of animals infected with bluetongue virus (BTV). There are several RT-PCR protocols published and several real-time RT-PCR (rtRT-PCR) commercial kits available on the market. Because Poland faced BTV-14 infection in 2012, different protocols were implemented in the country to confirm the RT-PCR results positive for this virus. The article presents a comparative study of several RT-PCR protocols and discusses their diagnostic reliability and applicability.Material and MethodsSix rtRT-PCR/RT-PCR protocols were compared for the laboratory diagnostic of fourteen BTV-14 isolates circulating in Poland in 2012–2014.ResultsAll 14 isolates were positive in the protocols of Shaw et al. (18), a commercial LSI NS3 kit, and Eschbaumer et al. (5). Four out of fourteen BTV-14 isolates gave positive results in Hoffmann’s 2 and 6 protocols and none of the 14 isolates yielded positive results in Maan et al. (8) method. Phylogenetic study of a short fragment of 450 nt of BTV segment 2 (258–696 positions) revealed 100% identity within Polish variants and with Russian and Spanish isolates.ConclusionThe paper points to the possible false negative results in the diagnosis of BTV infections depending on the protocol used.

Establishment and Application of a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Detection of Grapevine Fanleaf Virus

Grapevine degeneration in grapevines caused by Grapevine Fanleaf Virus (GFLV) has been documented in many viticulture regions worldwide. It is one of the major economically important virus diseases affecting the longevity of grapevines and reducing the fruit yield and fruit quality. To diminish the time required for some diagnostic assays, including reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and also double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture RT-PCR (IC-RT-PCR) protocol on the basis of GFLV genome were used and the reaction conditions were optimized for rapid detection of GFLV. In this regard, DAS-ELISA was employed first to validate the existence of the virus. All six RT-LAMP primers (i.e., F3, B3, FIP, BIP, LF and LB) together with RT-PCR primers (F and B) were designed on the basis of coat protein (CP) gene of GFLV. LAMP method, on the whole, had the following advantages over the other mentioned procedures: (I) fascinatingly, no need of RNA extraction; (II) no requirement of expensive and sophisticated tools for amplification and detection; (III) no post-amplification treatment of the amplicons; and (IV) a flexible and easy detection approach, which is visually detected by naked eyes using diverse visual dyes.

Identifying Diseases of Golden Jackals of Bangladesh Agricultural University Campus, Mymensingh, Bangladesh

Golden jackals is one of the semidomestic wild carnivors of the environment of Bangladesh Agricultural University (BAU) campus and now a days very often share spend their time with native dogs and cats in the gouse food wastes. This is not unusual for jackals to share diseases of dogs and cats. This study was aimed to identify important diseases of golden jackals and categorized to their zoonotic importance. A total of five apparently healthy golden jackals were collected from BAU campus and thorough postmortem examination was carried out during the period from July to December, 2010. Histopathological studies were conducted using routine Hematoxylin &amp; Eosin procedure. The existence of blood parasites were studied by Giemsa’s staining. Polymerase Chain Reaction (PCR) &amp; Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were adopted for the detection of tuberculosis (TB), canine distemper (CD) and avain influenza virus (AI). Other diseases investigated were liverfluke, whipworm, lungworm, mites and ascariasis. Out of five jackals examined, four were infected with heartworm Dirofilaria immitis. Gross examination of a Jackal at necropsy revealed nodular lesions in lungs and suspected as a case of TB. Acid fast staining and PCR protocol specific for TB could not detect mycobacterium in the nodular lungs lesions. Results of RT- PCR with the extracted RNA from liver showed amplification of 287bp fragment specific for CD viral infection in two cases. This is the first study in Bangladesh describing infection of CD in jackals. Jackals are scavanging in nature and reasonably AI could have present in jackals. RT-PCR protocol specific for matrix protein gene of AI viruses did not amplify any nucleic acid fragment. Results of this study showed that the golden jackals of BAU campus were infected with heartworm, lungworm, liverfluke, whipeworm, ascarids, mites and CD viruses. Extensive investigation is needed to explore the existance of few other important diseases of jackals including bovine and human TB, AI, leishmaniasis, rabies, Toxoplasmosis, Infectious canine hepatitis, Taeniasis and Canine hookworm infestation. heartworm, whipworm and mites have zoonotic importance, therefore, it needs to develop stretagy to prevent their future dessimination in human and other animals.DOI: http://dx.doi.org/10.3329/bjvm.v12i2.21295 Bangl. J. Vet. Med. (2014). 12 (2): 217-224

Molecular detection of Rift Valley fever virus in serum samples from selected areas of Tanzania

Rift Valley fever (RVF) is an acute mosquito-borne viral zoonotic disease affecting domestic animals and humans caused by the Rift Valley fever virus (RVFV). The virus belongs to the genus Phlebovirus of the family Bunyaviridae. The main aim of this study was to detect the presence of antibodies to RVFV as well as the virus in the serum samples that were collected from livestock during the 2006/2007 RVF outbreaks in different locations in Tanzania. Analysis of selected samples was done using a RVF-specific inhibition enzyme-linked immunosorbent assay (I-ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Genomic viral RNA was extracted directly from serum samples using a QIAamp Viral RNA Mini Kit (QIAGEN), and a one-step RT-PCR protocol was used to amplify the S segment of RVFV. Positive results were obtained in 39.5% (n = 200) samples using the RVF I-ELISA, and 17.6% (n = 108) of samples were positive by RT-PCR. I-ELISA detected 41 (38.7%), 32 (39.0%), and 6 (50.0%) positive results in cattle, goats, and sheep sera, respectively, whereas the RT-PCR detected 11 (0.2%), 7 (0.2%), and 1 (0.1%) positive results in cattle, goats, and sheep sera, respectively. These findings have demonstrated the presence of RVFV in Tanzania during the 2006/2007 RVF outbreaks. To our knowledge, this is the first report to detect RVFV in serum samples from domestic animals in Tanzania using PCR technique. Therefore, a detailed molecular study to characterize the virus from different geographical locations in order to establish the profile of strains circulating in the country and develop more effective and efficient control strategies should be done.

Simultaneous detection of Apple Chlorotic Leaf Spot Virus and Apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR

Apple Chlorotic Leaf Spot Virus (ACLSV; family Betaflexiviridae, genus Trichovirus) and Apple mosaic virus (ApMV; family Bromoviridae, genus Ilarvirus) are economically important viruses of apple (Malus×domestica Borkh.). A duplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of both ACLSV and ApMV along with an internal control (NADH dehydrogenase subunit 5 gene). The internal control was used to minimize the risk of getting false negative results. Specific primers were designed against coat protein gene of ApMV while for ACLSV & internal control previously reported primer sets were used. Firstly the uniplex RT-PCR assay was standardized for the detection of each virus independently. Then duplex RT-PCR protocol for simultaneous detection of ACLSV and ApMV was standardized by using primer pairs ACLSV-CP1F & ACLSV-CP1R and ApMV-CP1F & ApMV-CP5R, respectively, along with primer set for internal control. Robustness of the technique was further validated wherein; duplex RT-PCR was carried out to detect both viruses in crab apples, rootstocks and different popular cultivars of apple. Results reveal that the duplex RT-PCR assay has detection sensitivity as that of uniplex RT-PCR assay for respective viruses. This optimized duplex RT-PCR provides a simple, rapid, sensitive and convenient way for simultaneous detection of ACLSV & ApMV by reducing the time and cost of the supplies.

Simultaneous Detection and Survey of Three Rice Viruses in China.

Rice black-streaked dwarf virus, Rice stripe virus, and Southern rice black-streaked dwarf virus (SRBSDV) have been epidemic in large areas of China where rice is grown, causing significant losses of rice yield in recent years. These viral diseases sometimes occur in the same regions, and even in the same fields, making it difficult to detect and diagnose the viral pathogens. A set of primers specific to the genes encoding the capsid proteins of the three viruses were designed, and a multiple one-step reverse-transcription polymerase chain reaction protocol was developed. The method proved to be simple, rapid, and sensitive. It was used to detect the viruses in samples of rice, maize, small brown planthoppers, and white-backed planthoppers collected from different regions of China, showing that it is suitable for routine diagnosis. A simultaneous survey of the three viruses was further conducted by this method throughout Zhejiang Province, Eastern China. The results showed that both RBSDV and RSV had continued to spread and that the newly emerging virus, SRBSDV, was present in at least 27 counties or cities, suggesting that more effort is needed to monitor and control the threat from these three viral diseases.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Development of a combined canine distemper virus specific RT-PCR protocol for the differentiation of infected and vaccinated animals (DIVA) and genetic characterization of the hemagglutinin gene of seven Chinese strains demonstrated in dogs

A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China.

Multiplex RT-PCR assays for the simultaneous detection of both RNA and DNA viruses infecting cassava and the common occurrence of mixed infections by two cassava brown streak viruses in East Africa

Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocols were developed for the detection of cassava brown streak viruses (CBSVs) in single and mixed infections with cassava mosaic begomoviruses (CMBs) in a tropical crop plant, cassava (Manihot esculenta). CMBs contain ssDNA as their genome (genus Begomovirus, family Geminiviridae) while CBSVs are made up of positive sense ssRNA (genus Ipomovirus, family Potyviridae), and they cause the economically important cassava mosaic and cassava brown streak diseases, respectively, in sub-Saharan Africa. Diagnostic methodologies have long been available for CMBs but they are limited for CBSVs especially in mixed infections. In this study, the two CBSVs, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) occurring singly or in mixed infection with CMBs, African cassava mosaic virus and East African cassava mosaic virus were detected in a single RT-PCR using both previously described and newly designed virus-specific primers. These protocols were highly efficient for detecting CBSVs compared to the existing methods and have great potential to minimize sample handling and contamination. As well as improving the diagnosis of cassava viruses, the development of multiplex RT-PCR protocols have revealed the common occurrence of mixed infections by CBSV and CBSUV in cassava fields of Tanzania and Kenya, which was contrary to the common belief until recently that these two viruses have existed separately. These protocols have implications for diagnosis and epidemiological studies on cassava virus diseases in Eastern Africa.

Absence of &lt;emph type="ital"&gt;Helicobacter pylori&lt;/emph&gt; in Pediatric Adenoid Hyperplasia

To (1) develop a reverse transcription-polymerase chain reaction assay to determine whether Helicobacter pylori and/or other members of the Helicobacteraceae family are detected in hyperplastic adenoids of children and (2) critically analyze published polymerase chain reaction methods to ascertain whether false-positive detection of H pylori has been reported. Cohort study. Adenoid biopsy specimens (78 hyperplastic and 15 normal) were collected from children aged 2 to 10 years. Total RNA was extracted before reverse transcription of bacterial RNA using Helicobacteraceae-specific primer. A nested reverse transcription-polymerase chain reaction protocol was designed to detect all species of the Helicobacteraceae family. A piece of each biopsy specimen was examined histologically. Laryngopharyngeal reflux was suspected in 41% of the children (n=23) on the basis of the Reflux Symptom Index. No evidence of H pylori was found in any adenoid sample. Candidatus Wolinella africanus was the only Helicobacteraceae family member detected in 1 hyperplastic adenoid. Histologic examination identified very few bacterial organisms. Previous polymerase chain reaction findings may be the result of false-positive H pylori detection. Inflammation and enlargement of the adenoids is not likely due to ongoing bacterial infection arising from laryngopharyngeal reflux. We conclude that H pylori and other Helicobacteraceae family members are not major contributors to the development of hyperplastic adenoids in children.

Effective detection of human noroviruses in Hawaiian waters using enhanced RT-PCR methods

The current recreational water quality criteria using growth-based measurements of fecal indicator bacteria (FIB) concentration have their limitations for swimmer protection. To evaluate the possible use of enteric viruses as an improved indicator of human sewage contamination in recreational waters for enhanced health risk assessment, human norovirus (huNoV) was tested as a model in this study. To establish a highly sensitive protocol for effective huNoV detection in waters, 16 published and newly designed reverse transcription polymerase chain reaction (RT-PCR) primer pairs specific for huNoV genogroup I (GI) and genogroup II (GII) were comparatively evaluated side-by-side using single sources of huNoV RNA stock extracted from local clinical isolates. Under optimized conditions, these RT-PCR protocols shared a very different pattern of detection sensitivity for huNoV. The primer sets COG2F/COG2R and QNIF4/NV1LCR were determined to be the most sensitive ones for huNoV GII and GI, respectively, with up to 10 5- and 10 6-fold more sensitive as compared to other sets tested. These two sensitive protocols were validated by positive detection of huNoV in untreated and treated urban wastewater samples. In addition, these RT-PCR protocols enabled detection of the prevalence of huNoV in 5 (GI) and 10 (GII) of 16 recreational water samples collected around the island of O’ahu, which was confirmed by DNA sequencing and sequence analysis. Findings from this study support the possible use of enteric viral pathogens for environmental monitoring and argue the importance and essentiality for such monitoring activity to ensure a safe use of recreational waters.