Direct Reverse Transcriptase-polymerase Chain Reaction Research Articles

Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral lysis-amplification buffer prepared with IGEPAL-630

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.

Reno-Protective Effect of Realgar Nanoparticles on Lupus Nephritis of MRL/Lpr Mice through STAT1.

Realgar, an arsenic tetrasulfide compound, is a highly recognized traditional Chinese medicinal prescription that has been widely used to treat various diseases such as inflammatory diseases. However, there are still some problems in the clinical treatment of Realgar, such as large oral dose and high potential toxicity. To evaluate effects of Realgar nanoparticles on lupus nephritis (LN) in vivo in MRL/lpr mice. Ten-week mice were orally administered every day for eight consecutive weeks except the mice of normal model groups. The serum levels of anti-ds-DNA antibody IgG, IgM, IFN-γ, Creatinine (Cr), and blood urea nitrogen (BUN) were determined, and 24-hour urine protein was also measured. Renal inflammatory pathology analysis was assessed by hematoxylin-eosin (H&E) staining. The expression of phosphorylated signal transducer and activator of transcription 1 (p-STAT 1) and Janus Kinase 1 (JAK 1) in kidney tissue was determined by direct reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). The mice treated with Realgar nanoparticle in the high dose-treated (Realgar HD, 0.03 g/kg/d) group exhibited significantly reduced serum levels of anti-dsDNA (p<0.01), IgG (p<0.01), IgM (p<0.01), BUN (p<0.01), Cr (p<0.01), and inflammatory cytokine IFN-γ (p<0.01) as well as proteinuria (p<0.01) compared to the untreated model MRL/lpr mice. Additionally, high doses of Realgar nanoparticles significantly suppressed the phosphorylations of STAT 1(p<0.01) and the renal pathological changes. The study indicates that Realgar nanoparticles may be a potential agent to treat LN, and the down-regulated p-STAT1 expression suggests that it may be one of the LN treatment targets for Realgar nanoparticles.

A new, sensitive and efficient method for taxonomic placement in the Eriophyoidea and virus detection in individual eriophyoids.

Eriophyoids affect crops around the globe directly or indirectly as virus vectors. Eriophyoid systematics initiated over a century ago, yet more than 90% of their fauna remain undescribed. Morphological identification is challenging because of a limited number of traits, cryptic speciation and complex life cycle reported for many species in the group. Nucleic acids extraction for mite identification is challenging due to their microscopic size with researchers using pooled samples leading to polymorphisms and inconclusive results. Identification of mite virus vectors is a tiresome task that could be simplified with a protocol that allows for the detection of viruses in the individual specimen. This communication describes an innovative, highly efficient extraction and detection pipeline. Direct Reverse Transcriptase - Polymerase Chain Reaction (Drt-PCR) assays were implemented in the molecular identification of eriophyoids and detection of viruses present in their bodies. The reverse transcription step allows for amplification from a single mite or egg, as in addition to the genomic DNA, it incorporates the abundant transcripts of targeted genes, whereas it also allows for the amplification of viruses. This communication provides an efficient, sensitive and cost-effective alternative that can be implemented in pest identification and detection as well as biological and ecological studies.

The role of infectious bronchitis virus in respiratory and renal problems in broiler chickens

Broiler chickens in Egypt represent the main source of animal protein. Unfortunately, they usually are hit by many disease problems. Respiratory and renal affections are investigated in forty four broiler chicken flocks during 2014-2015 in three large Governorates; Sharkia, Dakahlia and Gharbia. The birds age ranged from 19-54days and suffured from general signs of illness, respiratory troubles with Whitish and greenish diarrhea. Mortality rates ranged from (0.5-30%). On necropsy, tracheitis, bronchitis and congestion of lungs were recorded in all flocks. Caseated plugs at tracheal bifurcation (29/44), fibrinous pericarditis, perihepatitis and air sacculitis(19/44) were also observed. Nepherosis-nephritis (22/44) and General congestion with hemorrhage on proventriculus (n=9) and cecal tonsil (n=4) were seen among affected birds. Chicken embryo inoculation with respiratory and renal tissue suspension revealed reduction in embryo size (n=9), death within 72hrs (n=24) while 5 flocks required successive blind passage to show reduction in embryo size up to 2cm compared to 5cm in negative control. The reverse transcriptase polymerase chain reaction (RT-PCR) was carried out on both original tissues and allantoic fluids using specific primers for S1 gene. Nine positive infectious bronchitis virus (IBV) were detected in original samples compared with only 2 in allantoic fluids of the same samples. It could be concluded that IBV is responsible on 20% of respiratory and renal troubles in broilers under investigation. The direct RT-PCR could be reliable tool for appropriate IBV diagnosis in chickens.

Isolation of Bovine Coronavirus (BCoV) in Vero Cell Line and its Confirmation by Direct FAT and RT-PCR

Bovine Coronavirus (BCoV) is widespread both in dairy and beef cattle throughout the world. The virus is one of the largest RNA virus and has specific tropism for intestinal and pulmonary epithelial cells. It is responsible for huge economic losses by causing winter dysentery in adult dairy cattle and respiratory and intestinal tract infections leading to pneumo-enteritis in young calves. Isolation of BCoV has been reported to be difficult. Studies regarding epidemiology, virus isolation and molecular detection from India are very few. In the present study Vero cell line was used for isolation of the BCoV from Enzyme Linked Immunosorbent Assay (ELISA) positive samples. Direct florescent antibody technique (dFAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to confirm the isolated virus strains at antigenic and genomic levels, respectively. Out of the 15 positive fecal samples, virus from only seven was able to infect vero cell line. Subsequently BCoV got adapted to the vero cell line upto three passages, which was confirmed both at genomic and antigenic levels by dFAT and RT-PCR testing. It can be concluded that vero cell line can be used for isolation of BCoV, however due to the enormous stain diversity of the virus it is possible that many stains can't grow and get adapt in this cell line. Further studies are required for isolation of different viral strains, finding the susceptible cell lines and also to confirm the variations among the BCoV isolates at antigenic/genomic levels.

Classification of IBV S1 Genotypes by Direct Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Relationship between Serotypes and Genotypes of Strains Isolated between 1998 and 2008 in Japan

In this study, we attempted to establish a simple detection method for classification of IBV S1 genotypes by direct reverse transcriptase-polymerase chain reaction (RT-PCR). Then, to evaluate the usefulness of the S1 genotype-specific RT-PCR, we examined the relationship between S1 genotypes and serotypes of IBV in Japan. Sequencing of the S1 genes of IBV and phylogenetic tree analysis were conducted. On the basis of the sequencing data of the S1 genotype samples, we determined primer sets specific for each genotype. Five vaccine strains in Japan as reference strains and 46 field isolates were classified into different genetic clusters by phylogenetic tree analysis (JP-1, JP-II, JP-III, Mass and 4/91) and were matched to the results of S1 genotype-specific RT-PCR. A cross virus-neutralizing test showed that the five vaccine strains in Japan exhibited different serotypes from each other. The concordance rate of the 46 field isolates between the S1 genotypes and serotypes was 65.2%. The present study indicates that genotype-specific RT-PCR could be a convenient and useful tool for determining IBV serotypes and could contribute to the control of IBV outbreaks in Japan.

Detection of noroviruses in fecal specimens by direct RT-PCR without RNA purification

Noroviruses are important human pathogens which cause epidemic acute viral gastroenteritis. Current techniques used for detection of noroviruses in fecal specimens involve multi-step viral RNA extraction and purification followed by reverse transcriptase-polymerase chain reaction (RT-PCR). This study demonstrates a method for easy detection of norovirus in fecal specimens, involving one-step RNA release and direct use of the released RNA for RT-PCR (direct RT-PCR). For one-step RNA release, a simple method was adopted based on addition of the sample treatment reagent from a commercialized Norovirus GI and GII RNA Detection Kit to suspended fecal specimens, followed by a brief heat treatment. The released RNA was then added directly to the RT mixture from the same kit. After reverse transcription and PCR, the product was detected by agarose gel electrophoresis. Direct RT-PCR was evaluated with 275 fecal specimens comprising 230 norovirus-positive and 45 norovirus-negative samples as assessed by real-time RT-PCR, considered to be the “gold standard” for norovirus detection. Direct RT-PCR was sufficiently specific and sensitive for norovirus detection, and eliminated the RNA extraction and purification step. Use of this method should facilitate detection of norovirus in fecal specimens and provide valuable information regarding the incidence of the virus. In addition, this method should be applicable for other RNA viruses.

Analysis of Coral Mucus as an Improved Medium for Detection of Enteric Microbes and for Determining Patterns of Sewage Contamination in Reef Environments

Traditional fecal indicator bacteria are often subject to a high degree of die-off and dilution in tropical marine waters, particularly in offshore areas such as coral reefs. Furthermore, these microbes are often not associated with human waste, and their presence may not be indicative of health risk. To address the offshore extent of wastewater contamination in the Florida Keys reef tract, we assayed coral surfaces for the presence of human-specific enteric viruses. The overlying water column and surface mucopolysaccharide (mucus) layers from scleractinian corals were sampled from three stations along a nearshore-to-offshore transect beginning at Long Key in the middle Florida Keys, USA. Samples were assayed for standard bacterial water quality indicators (fecal coliform bacteria and enterococci) and for human enteroviruses by direct reverse transcriptase-polymerase chain reaction (RT-PCR). The concentration of the bacterial indicators was greatest at the nearshore station in both the water column and corals, and decreased with distance from shore; no indicator bacteria were detected at the offshore station. Whereas human enteroviruses were not detected in any of the water column samples, they were detected in 50–80% of coral mucus samples at each station. These data provide evidence that human sewage is impacting the reef tract up to ~6.5 km from shore in the middle Florida Keys and that coral mucus is an efficient trap for viral markers associated with anthropogenic pollution.

Rapid concentration and detection of hepatitis A virus from lettuce and strawberries

Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 μl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (≤50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to ≤20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.

Reverse Transcriptase-Polymerase Chain Reaction Validation of 25 “Orphan” Genes from Escherichia coli K-12 MG1655

Despite the accumulation of sequence information sampling from a broad spectrum of phyla, newly sequenced genomes continue to reveal a high proportion (50%-30%) of "uncharacterized" genes, including a significant number of strictly "orphan" genes, i.e., putative open reading frames (ORFs) without any resemblance to previously determined protein-coding sequences. Most genes found in databases have only been predicted by computer methods and have never been experimentally validated. Although theoretical evolutionary arguments support the reality of genes when homologs are found in a variety of distant species, this is not the case for orphan genes. Here, we report the direct reverse transcriptase-polymerase chain reaction assay of 25 strictly orphan ORFs of Escherichia coli. Two growth conditions, exponential and stationary phases, were tested. Transcripts were identified for a total of 19 orphan genes, with 2 genes found to be expressed in only one of the two growth conditions. Our results suggest that a vast majority of E. coli ORFs presently annotated as "hypothetical" correspond to bona fide genes. By extension, this implies that randomly occurring "junk" ORFs have been actively counter selected during the evolution of the dense E. coli genome.

Identification of viable and non-viable Mycobacterium tuberculosis in mouse organs by directed RT-PCR for antigen 85B mRNA

Mycobacterium tuberculosis (MTB) the causative organism of tuberculosis can remain dormant as a non-culturable organism, reactivate and cause disease in man and animals. There is a need for proof of viability of such organisms in order to understand the process of reactivation. PCR for bacterial DNA cannot distinguish between viable and non-viable bacilli. We have tested a previously described two tube directed reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of mRNA of antigen 85B (Ag85B) of MTB that can distinguish between viable and non-viable organisms. Using a set of external and internal primers for Ag85B, a cDNA amplified product (216 bp) was seen among simulated samples containing only viable cfus at a sensitivity of >10 and <100 cfu/ml. Eucaryotic DNA rich normal mouse lung homogenate did not interfere among these samples. The method amplified the 216 bp product also among cfu positive tissues of naturally infected mice. Finally, in a mouse model of dormancy, direct RT-PCR detected a signal among multiple tissues that were negative for cfus and hence non-culturable. Ag85B is abundantly secreted by MTB and hyper-expressed under stress conditions. Thus the method to identify its mRNA message may be useful to detect viable but dormant bacteria.

Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies

The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.

Alpha-cardiac-like myosin heavy chain as an intermediate between MHCIIa and MHCI beta in transforming rabbit muscle.

To elucidate the sequence of myosin heavy chain (MHC) transitions in fast-to-slow transforming rabbit muscle, direct reverse transcriptase-polymerase chain reaction was applied for detecting mRNAs specific to five MHC isoforms in single fibers from control and low-frequency-stimulated tibialis anterior muscles. The detection of MHCIIb, MHCIId(x), MHCI alpha, and MHCI beta mRNAs was based on previously published methods. The RT-PCR assay for MHCIIa mRNA was based on the identification of a cDNA sequence in the 3'-region from which specific primers were derived. Comparisons between rat, rabbit, and human MHCIIa sequences revealed high degrees of sequence identities. MHC mRNA isoform patterns in single fibers from stimulated muscles showed hybrid fibers expressing the following combinations: MHCIId(x) + MHCIIa, MHCIId(x) + MHCIIa + MHCI alpha, MHCIId(x) + MHCIIa + MHCI alpha + MHCI beta, MHCIIa + MHCI alpha, MHCIIa + MHCI alpha + MHCI beta, and MHCI alpha + MHCI beta. The combination MHCIIa + MHCI beta without MHCI alpha was never seen. These coexpression patterns suggest that the fast-to-slow fiber transition results from sequential isoform expressions in the order MHCIId(x)--> MHCIIa-->MHCI alpha-->MHCI beta. The allocation of MHCI alpha between MHCIIa and MHCI beta seems to be in line with graded differences in sequence identity of the 3'-regions of these mRNA isoforms.

Quantitative analyses of myosin heavy-chain mRNA and protein isoforms in single fibers reveal a pronounced fiber heterogeneity in normal rabbit muscles.

A highly sensitive method of reverse-transcriptase polymerase chain reaction (RT-PCR) was established to study myosin heavy-chain (MHC) mRNA isoform expression in single fibers of rabbit limb muscles. In combination with myofibrillar adenosine triphosphatase histochemistry and electrophoretic separation of MHC protein isoforms in fragments of the same fibers, the direct RT-PCR method identified the pMHC20-40 and pMHC24-79 cDNA sequences as being specific to MHCIIb and MHCIId/x isoforms, respectively. In addition, a direct RT-PCR was established for determining relative amounts of MHC mRNA isoforms by using a sequence specific to alpha-skeletal actin as an endogenous reference. Analyses of large amounts of single fibers revealed an unexpected heterogeneity of the fast fiber population with regard to numerous fibers coexpressing MHCIIb and MHCIId/x. Based on quantitative RT-PCR, the percentages of MHCIIb/MHCIId hybrid fibers amounted to approximately 55% in the deep portion of gastrocnemius, to 43% in the adductor magnus, and to 12% in psoas muscle. Moreover, the two MHC mRNA isoforms were nonuniformly distributed along the fiber length. Qualitative RT-PCR detected even higher amounts of hybrid fibers in the three muscles. The percentages of hybrid fibers identified at the protein level were smaller in adductor magnus muscle (25%) and psoas muscle (5%), but equaled that of the mRNA analysis in gastrocnemius muscle (61%). The detection of high amounts of IIBD and IIDB fibers suggested that hybrid fibers represent functional elements within the fiber spectrum of normal muscles. Our observations on hybrid fibers reveal a heterogeneity within the fiber population of normal muscles that has not been realized to date.

Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

Isolation of black creek canal virus, a new hantavirus from Sigmodon hispidus in Florida.

Numerous rodents were trapped for serologic and virologic studies following the identification of a hantavirus pulmonary syndrome (HPS) case in Dade County, Florida. Cotton rats (Sigmodon hispidus) were the most frequently capture rodent and displayed the highest seroprevalence to a variety of hantavirus antigens. Hantavirus genome RNA was detected in all the seropositive cotton rats tested, using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A virus was isolated from tissues of two seropositive cotton rats by cultivation of lung and spleen homogenates on Vero E6 cells. Nucleotide sequence information obtained by direct RT-PCR and the serologic relationships of this virus with the other hantaviruses indicate that this virus, Black Creek Canal virus, represents a new hantavirus distinct from the previously known serotypes.

Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.

Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptasepolymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 µL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.