Reverse Transcription-polymerase Chain Reaction Kit Research Articles

The Limited Sensitivity of Chest Computed Tomography Relative to Reverse Transcription Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus-2 Infection: A Systematic Review on COVID-19 Diagnostics.

ObjectivesSeveral studies suggest the sensitivity of chest computed tomography (CT) is far greater than that of reverse transcription polymerase chain reaction (RT-PCR) in diagnosing COVID-19 patients, and therefore, CT should be included as a primary diagnostic tool. This systematic review aims to stratify studies as high or low risk of bias to determine the true sensitivity of CT for severe acute respiratory syndrome coronavirus-2 infection according to the unbiased (low risk) studies, a topic of particular importance given the insufficient quantity of RT-PCR kits in many countries. We focus on sensitivity as that is the chief advantage perceived of CT.Materials and MethodsThis systematic review involved searching the PubMed and Google Scholar databases for articles conducted and published between January 1 and April 15, 2020. The quality assessment tool QUADAS-2 was used to stratify studies according to their risk of bias, and exclusion criteria included not providing the information deemed relevant for such a stratification, such as not indicating if the patients were symptomatic or asymptomatic, or identifying the source of the specimen for the reference standard, RT-PCR (eg, nasal, oropharyngeal, etc). Sensitivity values were then extracted, and random effects meta-analyses were performed.ResultsOf 641 search results, 37 studies (n = 9610 patients) were included in the analysis. The mean sensitivity of RT-PCR for COVID-19 reported by the biased studies was 70% (n = 5409/7 studies; 95% confidence interval [CI], 43–97; I2 = 99.1%), compared with 78% by unbiased studies (n = 534/4 studies; 95% CI, 69–87, I2 = 89.9%). For chest CT, the mean sensitivity reported by biased studies was 94% (n = 3371 patients/24 studies; 95% CI, 92–96; I2 = 93.1%), compared with 75% by unbiased studies (n = 957/10 studies; 95% CI, 67–83; I2 = 89.5%).ConclusionsThe difference between the sensitivities of CT and RT-PCR for severe acute respiratory syndrome coronavirus-2 infection is lower than previously thought, as after stratifying the studies, the true sensitivity for CT based on the unbiased studies is limited.

A Direct from Blood/Plasma Reverse Transcription-Polymerase Chain Reaction for Dengue Virus Detection in Point-of-Care Settings.

Infection with dengue virus (DENV) is widespread across tropical regions and can result in severe disease. Early diagnosis is important both for patient management and to differentiate infections that present with similar symptoms, such as malaria, chikungunya, and Zika. Rapid diagnostic tests that are used presently for point-of-care detection of DENV antigens lack the sensitivity of molecular diagnostics that detect viral RNA. However, no molecular diagnostic test for DENV is available for use in field settings. In this study, we developed and validated a reverse transcription-polymerase chain reaction (RT-PCR) for the detection of DENV adapted for use in field settings. Reverse transcription-polymerase chain reaction was performed directly from plasma samples without RNA extraction. The assay detected all four serotypes of DENV spiked into blood or plasma. Our RT-PCR does not cross-react with pathogens that cause symptoms that overlap with dengue infection. The test performed equally well in a conventional laboratory qPCR instrument and a small, low-cost portable instrument that can be used in a field setting. The lower limit of detection for the assay was 1 × 104 genome copy equivalents/mL in blood. Finally, we validated our test using 126 archived patient samples. The sensitivity of our RT-PCR was 76.7% (95% CI: 65.8-87.9%) on the conventional instrument, and 78.3% (95% CI: 65.8-87.9%) on the field instrument, when compared with the RealStar Dengue RT-PCR Kit 2.0. The molecular test described here is user-friendly, low-cost, and can be used in regions with limited laboratory capabilities.

First Report of Chrysanthemum Stem Blight and Dieback Caused by Tomato spotted wilt virus in China

Cultivated chrysanthemum (Chrysanthemum morifolium) is a popular flower species from China, where it is widely planted for use as tea and in medicines, as well as for ornamental purposes. In July 2016, a serious stem necrotic disease was observed on chrysanthemum in a commercial greenhouse in Beijing, China. Most chrysanthemum varieties were infected, with a total disease incidence above 50%, rising to over 90% in some sensitive cultivars. Infected chrysanthemum plants exhibited two main symptoms: (i) irregular chlorotic spots on the upper leaves with shortened internodes (cultivar Silvia), or (ii) discontiguous black streaks on stems near the base of the upper petioles, followed by eventual dieback (cultivars Dante, Radost, Stellini, and Olive). These symptoms are very similar to those of chrysanthemum infected with the tospoviruses Tomato spotted wilt virus (TSWV) or Chrysanthemum stem necrotic virus (CSNV). To identify the pathogen, infected chrysanthemum leaves were analyzed using reverse-transcription polymerase chain reaction (RT-PCR) with tospovirus universal primers (Hassani-Mehraban et al. 2016). After extracting the total RNA with a Plant RNA kit (Tiangen, Beijing, China), RT-PCR was performed using an RT-PCR kit (Vazyme, Nanjing, China), with infected chrysanthemum showing black streaks on stems as positive controls and virus-free plantlets as negative controls. RT-PCR products were bidirectionally sequenced by Sangon Biotech (Shanghai, China) and aligned in DNAstar. Three clones of 10 infected leaf samples (two of each cultivar) were collected from different sections of the greenhouse and sequenced, revealing high sequence similarity with fewer than 2-bp difference between them. The most conserved sequence, isolated from sample BLZ01, was deposited in GenBank under accession number MF196915. This sequence was 99% identical to TSWV S RNA segments in GenBank and distinctly different from CSNV RNAs, indicating that the devastating disease was caused by TSWV, not CSNV. This diagnosis was further confirmed using commercial ELISA kits (Luyuan Bode Biological, Beijing, China), which revealed TSWV but not CSNV in all symptomatic tissue. Additional RT-PCRs were performed using TSWV-specific (Chung et al. 2006; Qiu et al. 1998) and CSNV-specific (Takeshita et al. 2011) primers complementing the RNA-dependence RNA polymerase gene (L RNA), Gn/Gc gene (M RNA), and nucleocapsid protein gene (NP, S RNA), respectively, and the results confirmed those of the ELISA. Finally, the virus isolated from BLZ01 was mechanically transmitted from infected cultivar Olive collected from the original greenhouse to 1-month-old virus-free chrysanthemum seedlings using chilled 0.01 M phosphate buffer (pH 7.2) containing 0.1% sodium sulfite. After 2 weeks, most inoculated plants (13 of 18) exhibited localized necrotic spots and black streaks on their stems. RT-PCR and sequencing revealed that the partial sequence of the TSWV NP gene from the inoculated chrysanthemum was identical to the virus from the greenhouse materials. TSWV has spread and is causing epidemics on chrysanthemum in many countries. To our knowledge, this is the first report of TSWV detected on chrysanthemum in China, and this article will act as a warning to prevent the spread of TSWV in Beijing.

Bead-based suspension array for simultaneous differential detection of five major swine viruses.

A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/μL (corresponding to 0.5-6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.

Diagnosis of human parechovirus infections of the central nervous system with a commercial real-time reverse transcription–polymerase chain reaction kit and direct genotyping of cerebrospinal fluid specimens

We screened 100 cerebrospinal fluid specimens for the human parechoviruses (HPeV) genome with the commercial parechovirus r-gene™ kit, which allows results to be available in a clinically relevant time frame. HPeV infection was diagnosed in 4 infants (<4 months) and all genotyped viruses were HPeV3.

Heightened Induction of Proapoptotic Signals in Response to Endoplasmic Reticulum Stress in Primary Fibroblasts from a Mouse Model of Longevity

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58(IPK) and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.

Development, optimization, and validation of a Classical swine fever virus real-time reverse transcription polymerase chain reaction assay

Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a well-characterized assay that can deliver a rapid, accurate diagnosis prior to the onset of clinical signs. A real-time fluorogenic-probe hydrolysis (TaqMan) reverse transcription polymerase chain reaction (RT-PCR) for CSF was developed by the United States Department of Agriculture (USDA) at the Plum Island Animal Disease Center (CSF PIADC assay) and evaluated for analytical and diagnostic sensitivity and specificity. A well-characterized panel including Classical swine fever virus (CSFV), Bovine viral diarrhea virus (BVDV), and Border disease virus (BDV) isolates was utilized in initial feasibility and optimization studies. The assay was initially designed and validated for use on the ABI 7900HT using the Qiagen QuantiTect® Probe RT-PCR chemistry. However, demonstrating equivalency with multiple one-step RT-PCR chemistries and PCR platforms increased the versatility of the assay. Limit of detection experiments indicated that the Qiagen QuantiTect® Multiplex (NoROX) and the Invitrogen SuperScript® III RT-PCR kits were consistently the most sensitive one-step chemistries for use with the CSF PIADC primer/probe set. Analytical sensitivity of the CSF PIADC assay ranged from <1-2.95 log(10) TCID(50)/ml on both the ABI 7900HT and ABI 7500 platforms. The CSF PIADC assay had 100% diagnostic sensitivity and specificity when tested on a panel of 152 clinical samples from the Dominican Republic and Colombia. The ability to perform this newly developed assay in 96-well formats provides an increased level of versatility for use in CSF surveillance programs.

P85α Regulates Osteoblast Differentiation by Cross-talking with the MAPK Pathway

Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.

Development and field evaluation of a nested RT-PCR kit for detecting Japanese encephalitis virus in mosquitoes

A novel nested reverse transcription-polymerase chain reaction (RT-PCR)-based kit is described for detecting Japanese encephalitis virus (JEV), especially for genotype 1 and 3 strains. The assay consists of a first round RT-PCR and a subsequent nested PCR amplification. It has unique features such as the use of a premix system in which all reagents are lyophilized in reaction tubes and the inclusion of control RNA in each reaction to monitor false negative results. In addition, an automatic tissue homogenizer and a RNA extraction system are used concurrently for assay standardization and increasing throughput. The assay using the kit proved specific for JEV with no amplification of other JEV-related flaviviruses. The detection limits were approximately 0.1 PFU/ml and 1 PFU/ml for JEV genotypes 1 and 3, respectively. The assay protocol has been validated in large-scale field trials in South Korea during the 2008–2009 surveillance seasons. Nineteen of 1136 pools of mosquitoes (54,583 mosquitoes total) were identified as JEV positive. This nested RT-PCR kit combined with control RNA and an automatic RNA extraction system should be suitable for routine JEV surveillance programs.

First report of Barley yellow mosaic virus infecting barley in Poland

Barley yellow mosaic virus (BaYMV) is a dangerous pathogen of winter barley in Europe and Japan, transmitted by the plasmodiophorid vector Polymyxa graminis (Adams, 1996). No data on the occurrence of barley yellow mosaic viruses were recorded in Poland until spring 2008. First disease symptoms appeared in at least 50% of winter barley fields by late March, affecting mainly cv. Vanessa in Lower Silesia region. These were associated with mosaic and leaf yellowing, including longitudinal chlorotic flecks on younger leaves of some plants, decreased growth, and irregular yellow patches in the fields. Leaves of barley plants with symptoms were collected during April and May, and tested by ELISA using antibodies against BaYMV (Loewe), yielding positive results with 135 out of 352 samples. The OD values ranged from three to 10 times higher than the negative controls. Bymovirus-like flexuous particles, about 700 nm long, were observed under the electron microscope (Philips EM-201) in leaf extracts only from samples from symptom-bearing leaves. Fourteen plant samples, found to be ELISA positive for BaYMV were indexed by reverse transcription‐polymerase chain reaction (RT-PCR). Total RNA was isolated from 100‐120 mg fresh plant tissue using RNeasy Mini Kit (Qiagen), reverse transcribed and then amplified by PCR using a one step RT-PCR kit (Qiagen) with primers BaYMV-F and BaYMV-R (Vaianopoulos et al ., 2003). PCR products of the expected size (346bp) observed in nine samples were separated electrophoretically in a 1% agarose gel. The consensus sequence from two amplicons was deposited in GenBank (FJ436987). The BaYMV partial coat protein sequence showed the highest identity (100%) with that of BaYMV from UK (AJ515485). The virus was easily propagated in glasshouse conditions by soil-transmission experiments. The identification of BaYMV in Poland is an important indication for barley growers to cultivate only resistant winter barley cultivars in the regions of the disease incidence.

First Report of Cucurbit aphid-borne yellows virus in Tunisia Causing Yellows on Five Cucurbitacious Species.

Viruses, distributed worldwide on cucurbits, cause severe damage to crops. Virus surveys in 2003 and 2004 were made in all the major cucurbit-growing areas in Tunisia. Large populations of aphids (Aphis gossypii Glover) and severe yellowing symptoms of older leaves of cucurbits were observed in outdoor and under plastic-tunnel cultivation, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae). Leaf samples collected from symptomatic and asymptomatic plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita pepo L.), watermelon (Citrullus lanatus L.), and ware cucurbit (Ecballium elaterium L. T. Richard) were screened for the presence of CABYV using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Reference isolate, CABYV-N (GenBank Accession No. X76931) was provided by H. Lecoq (INRA-Monfavet Cedex, France). Sample extracts from fresh leaf tissues were tested using ELISA with an antiserum prepared against this isolate. In addition, total RNA was extracted from fresh leaf tissues according to the technique of Celix et al. (2) using the Titan RT-PCR kit from Roche Diagnostics (Penzberg, Germany). Forward primer (5'-GAGGCGAAGGCGAAGAAATC-3') and reverse primer (5'-TCTGGACCTGGCACTTGATG-3') were designed with the available sequence of the reference isolate. ELISA tests demonstrated that 91 plants were positive among 160 plants tested with severe yellowing symptoms. All asymptomatic plants were negative. RT-PCR results yielded an expected 550-bp product that was amplified from the reference isolate. Of the 160 plants tested using ELISA, 106 plants were screened with RT-PCR including the 91 plants that were positive in ELISA. These 91 plants also were positive after RT-PCR amplification as were 12 more plants. This demonstrated that the RT-PCR test is more sensitive. No amplicons were produced from extracts of asymptomatic plants, RNA preparations of Cucurbit yellow stunting disorder virus (CYSDV), or Beet pseudo yellows virus (BPYV) positive controls provided by B. Falk (University of California, Davis). CYSDV and BPYV can induce similar yellowing symptoms in cucurbits. The results of the ELISA and RT-PCR tests showed that CABYV is widely distributed on five cucurbit species in the major growing areas of Tunisia including the northern, Sahel, central, and southern regions where it was detected, respectively, in 10 of 25, 11 of 21, 24 of 37, and 58 of 77 samples tested. CABYV was detected at the rates of 63 of 72 on melon, 10 of 21 on cucumber, 17 of 24 on squash, 10 of 25 on watermelon, and 3 of 18 on ware cucurbit. CABYV also seems to be widespread throughout the Mediterranean Basin (1,3,4), but to our knowledge, this is the first report of the occurrence of CABYV in Tunisia on different species of cucurbit and ware cucurbit.

Occurrence of hepatitis A virus genotype III in Germany requires the adaptation of commercially available diagnostic test systems

A blood donation, obtained in 2003 in Germany during the preseroconversion diagnostic window period of a hepatitis A virus (HAV) infection, tested HAV-negative by commercially available HAV reverse transcription-polymerase chain reaction (RT-PCR) detection assays. The virus responsible for this infection was identified as HAV genotype IIIA by characterization of the nearly complete genome sequence. Thereby, this HAV variant, which was named strain HMH, was detected in Germany for the first time. Because the commercially available HAV RNA detection systems failed to detect this genotype, a real-time RT-PCR kit was developed that allows quantification and detection of all HAV genotypes. The first nearly full-length nucleotide sequence so far available for HAV genotype IIIA is also provided. This case demonstrates that owing to the genetic variability of HAV, constant monitoring and adaptation of the diagnostic nucleic acid assays are required to guarantee the safety of blood and blood products.

Histamine Induces Melanogenesis and Morphologic Changes by Protein Kinase A Activation via H2 Receptors in Human Normal Melanocytes

Hyperpigmentation frequently accompanies chronic or acute inflammation. A number of inflammatory mediators have been shown to stimulate melanin synthesis in human melanocytes. Although histamine is ubiquitous as an inflammatory factor, its involvement in pigmentation remains obscure. In this work, we examined the effects of histamine on cultured human melanocytes. Treatment of human melanocytes with 0.1-10 microM histamine evoked morphologic changes and increases in tyrosinase activity. The concomitant increases in melanin content of the histamine-treated melanocytes indicated an elevation of melanin synthesis by tyrosinase activation. These stimulatory effects of histamine were completely inhibited by an H2 antagonist, famotidine, whereas H1 and H3 antagonists had no inhibitory effect whatsoever. In addition, an H2 agonist, dimaprit, induced the same degree of melanogenesis as histamine at concentrations of 0.1-10 microM. We observed an increase in the intracellular cAMP contents of human melanocytes induced by histamine via the H2 receptors. We know that this cAMP accumulation and subsequent protein kinase A activation plays a critical role in histamine-induced melanogenesis, because a specific protein kinase A inhibitor, H-89, completely suppressed these stimulatory effects of histamine, and because dibutylic cAMP, a specific protein kinase A activator, stimulated human melanocytes as potently as histamine. Taken together, we show here that histamine induces melanogenesis of human cultured melanocytes by protein kinase A activation via H2 receptors.