Real-time PCR Detection Research Articles

Utility of ultra-rapid real-time PCR for detection and prevalence of Rickettsia spp. in ticks

BackgroundRickettsia spp. are important tick-borne pathogens that cause various human and animal diseases worldwide. A tool for rapid and accurate detection of the pathogens from its vectors is necessary for prevention of Rickettsioses propagation in humans and animals, which are infested by ticks. Therefore, this study was conducted to evaluate a molecular tool, ultra-rapid real-time PCR (UR-qPCR), for rapid and accurate detection of Rickettsia spp. from 5644 ticks in 408 pools collected from livestock and their surrounding environments in Gangwon and Jeju province in South Korea.ResultsThe UR-qPCR of Rickettsia DNA showed a limit of detection of 2.72 × 101 copies of Rickettsia DNA and no cross reaction with other tick-borne pathogens, namely Anaplasma phagocytophilum, Ehrlichia chaffeensis, E. canis, Toxoplasma gondii, and Borrelia burgdorferi. In addition, the PCR assay also showed possibility of various Rickettsia species detection including R. monacensis, “Candidatus R. longicornii”, R. japonica, R. roultii, and R. tamurae. The collected ticks were identified with major species belonged to Haemaphysalis longicornis (81.62%), followed by H. flava (15.19%), and Ixodes nipponensis (3.19%). Rickettsia detection from tick samples using the UR-qPCR showed that the minimum infection rate (MIR) of Rickettsia in collected ticks was 1.24‰ and that all positive pools contained H. longicornis, equal to the MIR of 1.39‰ of this species. Additionally, MIR of Rickettsia spp. detected in ticks collected in Gangwon and Jeju was 1.53‰ and 0.84‰, respectively. Furthermore, the sequencing results of the 17 kDa protein antigen gene and ompA gene showed that Rickettsia spp. sequences from all pools were related to “Candidatus R. longicornii” and “Candidatus R. jingxinensis”.ConclusionsThe UR-qPCR system was demonstrated to be useful tool for accurate and rapid detection of Rickettsia from its vector, ixodid ticks, within 20 min. The data on Rickettsia spp. in ticks detected in this study provide useful information on the distribution of Rickettsia in previously unstudied Korean provinces, which are important for the prevention and control of the spread of rickettsioses in both animals and humans in the country.

Real-time PCR for detection and quantification of C. gloeosporioides s.l. growth in Stylosanthes and Arabidopsis

Colletotrichum gloeosporioides sensu lato (s.l.) is one of the most economically and scientifically important fungal pathogens, causing serious diseases in tropical and subtropical region. The detection of C. gloeosporioides s.l. is of importance for disease control. In this study, an ACT-based real-time PCR assay was developed for quantification of C. gloeosporioides s.l., which reliably detected as little as 100 fg genomic DNA, 100 copies of target DNA and 20 conidia. This method could recognize all four tested C. gloeosporioides s.l. isolates, while no amplification was observed in other Colletotrichum species and Botrytis cinerea, indicating the specificity of this assay. Detection and quantification of C. gloeosporioides s.l. was demonstrated in artificially and naturally infected host leaves. First, the real-time PCR analysis was performed using leaf samples collected at different time points post inoculation to monitor the growth of C. gloeosporioides s.l. over time. Secondly, the method was used to compare the resistance in two Stylosanthes cultivars and two Arabidopsis cultivars. Finally, naturally infected and symptomless leaves of Stylosanthes in the fields were tested by the real-time PCR method. Overall, the real-time PCR assay could allow the detection at early symptomless phase of infection; and the results was well correlated with microscopic observation and later disease symptoms. Therefore, our studies have provided a rapid and effective detection method for studying the plant-Colletotrichum interaction as well as disease prediction and control.

An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products.

The growing demand for and supply of meat and meat products has led to a proportional increase in cases of meat adulteration. Adulterated meat poses serious economic and health consequences globally. Current laboratory methods for meat species identification require specialized equipment with limited field applications. This study developed an inexpensive, point-of-care Loop-Mediated Isothermal Amplification (LAMP)-CRISPR/Cas12a colorimetric assay to detect meat species using a Texas Red-labelled single-strand (ssDNA) reporter. As low as 1.0 pg/µL of the porcine NADH4, the chicken NADH dehydrogenase subunit 2 (ND2) and the duck D-loop genes was detectable under white, blue and ultraviolet light. The test turnaround time from DNA extraction to visualization was approximately 40 min. The assay accurately detected pure and mixed-meat products in the laboratory (n = 15) and during a pilot point-of-care test (n = 8) in a food processing factory. The results are 100% reproducible using lateral flow detection strips and the real-time PCR detection instrument. This technology is fully deployable and usable in any standard room. Thus, our study demonstrates that this method is a straightforward, specific, sensitive, point-of-care test (POCT) adaptable to various outlets such as customs, quarantine units and meat import/export departments.

Assessment of SARS-CoV-2 tests costs and reimbursement tariffs readjustments during the COVID-19 pandemic.

While laboratories have been facing limited supplies of reagents for diagnostic tests throughout the course of the COVID-19 pandemic, national and international health plans, as well as billing costs, have been constantly adjusted in order to optimize the use of resources. We aimed to assess the impact of SARS-CoV-2 test costs and reimbursement tariff adjustments on diagnostic strategies in Switzerland to determine the advantages and disadvantages of different costs and resource saving plans. We specifically assessed the cost of diagnostic SARS-COV-2 RT-PCR using five different approaches: i) in-house platform, ii) cobas 6800® (Roche, Basel, Switzerland), iii) GeneXpert® SARS-CoV-2 test (Cepheid, Sunnyvale, CA, USA), iv) VIASURE SARS-CoV-2 (N1 + N2) Real-Time PCR Detection Kit for BD MAX™ (Becton Dickinson, Franklin Lake, NJ, USA), v) cobas® Liat® SARS-CoV-2 & Influenza A/B (Roche, Basel, Switzerland). We compared these costs to the evolution of the reimbursement tariffs. The cost of a single RT-PCR test varied greatly (as did the volume of tests performed), ranging from as high as 180 CHF per test at the beginning of the pandemic (February to April 2020) to as low as 82 CHF per test at the end of 2020. Depending on the time period within the pandemic, higher costs did not necessarily mean greater benefits for the laboratories. The costs of molecular reagents for rapid tests were higher than of those for classic RT-PCR platforms, but the rapid tests had reduced turnaround times (TATs), thus improving patient care and enabling more efficient implementation of isolation measures, as well as reducing the burden of possible nosocomial infections. At the same time, there were periods when the production or distribution of these reagents was insufficient, and only the use of several different molecular platforms allowed us to sustain the high number of tests requested. Cost-saving plans need to be thoroughly assessed and constantly adjusted according to the epidemiological situation, the clinical context and the national resources in order to always guarantee that the highest performing diagnostic solutions are available. Not all cost-saving strategies guarantee good analytical performance.

Enzalutamide-resistant related lncRNA NONHSAT210528 promotes the proliferation and invasion of prostate cancer.

BackgroundAs a new-generation androgen-receptor antagonist, enzalutamide is a first-choice drug for advanced prostate cancer (PCa) patients. However, secondary resistance to enzalutamide poses a new challenge in the treatment of cancer. Long non-coding RNA (lncRNA) regulates cell function through many levels and mechanisms, and also plays an important role in the biological behaviors of tumors.MethodsLncRNA microarrays were used to detect enzalutamide-resistant related lncRNA in Enzalutamide-resistant C4-2 (C4-2 ENZ-R) cells and corresponding parent cells. Cell Counting Kit 8, flow cytometry, and transwell assays were used to test the effect of lncRNA NONHSAT210528 on the function of PCa cells. RNA pulls down and the luciferase report gene was used to detect the competitive endogenous RNA (ceRNA) mechanism. The culture supernatant of C4-2 and C4-2b cells was transferred to the lower chamber for transwell assay of human umbilical endothelial cells (HUVECs).ResultsThe lncRNA microarray analysis showed that there were significant differences in the expression of many lncRNAs between the C4-2 ENZ-R and C4-2 cells. The real-time polymerase chain reaction (PCR) detection showed that the expression of lncRNA NONHSAT210528 was significantly higher in the C4-2 ENZ-R cells than the C4-2 cells. The Transwell assays showed that lncRNA NONHSAT210528 overexpression increased the invasion of the C4-2 and C4-2b cells. The cell-wound scratch and the transwell assays showed that the culture supernatant of C4-2 and C4-2b cells with overexpressed lncRNA NONHSAT210528 promoted the migration and invasion of HUVECs. Furthermore, lncRNA NONHSAT210528 regulated the expression of YOD1 dependent on miR-21.ConclusionsEnzalutamide-resistant related lncRNA NONHSAT210528 appears to promote the proliferation and invasion of PCa cells by functioning as a ceRNA and regulating the miR-21-5p/YOD1 signal pathway.

First Report of TTSuV1 in Domestic Swiss Pigs.

Serum prevalence of Torque teno sus viruses (TTSuV1 and k2; family Anelloviridae) is known to be high in the porcine population worldwide but pathogenesis and associated pathomorphological lesions remain to be elucidated. In this study, quantitative real-time PCR for detection of TTSuV1 was performed in 101 porcine samples of brain tissue, with animals showing inflammatory lesions or no histological changes. Additionally, a pathomorphological and immunohistochemical characterization of possible lesions was carried out. Selected cases were screened by TTSuV1 in situ hybridization. Furthermore, TTSuV1 quantitative real-time PCR in splenic and pulmonary tissue and in situ hybridization (ISH) in spleen, lungs, mesenteric lymph node, heart, kidney, and liver were performed in 22 animals. TTSuV1 was detected by PCR not only in spleen and lung but also in brain tissue (71.3%); however, in general, spleen and lung tissue displayed lower Ct values than the brain. Positive TTSuV1 results were frequently associated with the morphological diagnosis of non-suppurative encephalitis. Single TTSuV1-positive lymphocytes were detected by ISH in the brain but also in lungs, spleen, mesenteric lymph node and in two cases of non-suppurative myocarditis. A pathogenetic role of a TTSuV1 infection as a co-factor for non-suppurative encephalitides cannot be ruled out.

Detection of Human Herpes Virus-6 in saliva of Patients with Bell's palsy

Background: Bell’s palsy is unilateral peripheral paralysis of the seventh cranial nerve, several mechanisms have been proposed in the pathogenesis of this disease, among the viral infections specially herpes virus's family including roseola viruses that have been detected in patients' saliva.
 Method: A case-control study conducted on Bell’s palsy patients at al-Imamein Al-kadhimein Medical City throughout the period from March 2019 to November 2019. Saliva samples were taken from 50 patients (18-55) years of age with early stages of Bell's palsy and from 50 apparently healthy and age and sex matched volunteers as control. Viral DNA was extracted from the saliva and then real time PCR for detection and quantification of HHV6 DNA in these patients
 Results: Saliva samples taken from 28 patients in the Bell’s palsy patients (56%) tested positive for HHV6, as vs. to just 8 (16%) in the healthy group(control) (P= 0.028). There was increase amount of HHV6 load in patients compare to control group (p=0.001). Regarding the demographic values, an important difference in the detection rate for Human herpes virus-6 between male (64%) and females (35%) (P= 0.02). There was significant correlation between age and grading (p =0.01), as increase age correlate with high grading, also between viral load of HH6 and grading as increase viral load of the virus correlate with high grading (p = 0.001).
 Conclusion: HHV-6 virus might have a potential role in the pathogenesis of Bell’s palsy.

Head-to-head comparison of the diagnostic value of five tests for constrictive tuberculous pericarditis: Five tests for constrictive TBP

BackgroundThe diagnosis of constrictive tuberculous pericarditis (TBP) remains challenging. This study aimed to evaluate 5 tests (acid-fast bacilli [AFB] smear, Mycobacterium tuberculosis [MTB] culture, Xpert MTB/RIF assay, CapitalBio Mycobacterium real-time PCR detection assay [CapitalBio assay], and pathology) for constrictive TBP using pericardial tissue. MethodsWe reviewed the case histories of patients with suspected constrictive TBP. We analyzed the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of these assays. ResultsA total of 69 patients were included. The sensitivity, specificity, PPV, NPV, and AUC of AFB smear were 7.3%, 100.0%, 100.0%, 21.5%, and 0.54, respectively; those of culture were 23.6%, 100.0%, 100.0%, 25.0%, and 0.62, respectively; those of Xpert MTB/RIF were 52.7%, 100.0%, 100.0%, 35.0%, and 0.76, respectively; those of CapitalBio assay were 50.9%, 100.0%, 100.0%, 34.2%, and 0.75, respectively; and those of pathology were 92.7%, 92.9%, 98.1%, 76.5%, and 0.93, respectively. ConclusionsThe validity of AFB smear and MTB culture remains low. Nucleic acid amplification tests can provide diagnostic efficacy for TBP but only moderately. The CapitalBio assay and Xpert MTB/RIF were considered similar for diagnosing TBP. Pathology showed the best diagnostic accuracy among the 5 tests.

Combining a Universal Capture Ligand and Pan-Serotype Monoclonal Antibody to Develop a Pan-Serotype Lateral Flow Strip Test for Foot-and-Mouth Disease Virus Detection.

Foot-and-mouth disease virus (FMDV) causes FMD, a highly contagious disease of cloven-hoofed animals including cattle, goats, pigs and sheep. Rapid detection of FMDV is critical to limit the devastating economic losses due to FMD. Current laboratory methods for FMDV detection such as virus isolation, real-time reverse transcription PCR and antigen detection enzyme-linked immunosorbent assay (AgELISA) are labor-intensive, requiring trained personnel and specialized equipment. We present the development and validation of a pan-serotype lateral flow strip test (LFST) that uses recombinant bovine integrin αvβ6 as a universal capture ligand and a pan-serotype monoclonal antibody (mAb) to detect FMDV. The LFST detected all seven FMDV serotypes, where the diagnostic sensitivity was comparable to the AgELISA, and the diagnostic specificity was 100% without cross-reactivity to other viruses causing vesicular disease in livestock. This rapid test will be useful for on-site FMDV detection, as well as in laboratories in endemic countries where laboratory resources are limited.

Identification of matB used as an endogenous reference gene for the qualitative and real-time quantitative polymerase chain reaction detection of Lentinus edodes.

Lentinus edodes is a fungus with rich nutritional value and good medicinal value and has accordingly become a substitute for other expensive wild edible mushrooms. In this study, for the first time, the matB gene was selected as an endogenous reference gene of L. edodes and identified as the species‐specific gene. The matB genes of L. edodes and 18 non‐L. edodes species were determined by qualitative polymerase chain reaction (PCR), but no amplification was found in non‐L. edodes species. In SYBR Green quantitative PCR analysis, the detection limit was as low as 16 pg/µl of DNA template. All of these experiments indicated that the matB gene is an ideal reference gene and can detect L. edodes material through qualitative and quantitative PCR assays. It also provides a convenient and accurate approach for the detection of L. edodes products and the adulteration in wild edible mushroom products.

Luciferase Immunosorbent Assay Based on Multiple E Antigens for the Detection of Chikungunya Virus-Specific IgG Antibodies.

ABSTRACTIn recent years, the chikungunya virus (CHIKV) has continued to spread from local epidemics to nonnative habitats until eventually reaching pandemic status. Nonendemic areas such as China have also emerged as potential epidemic areas of CHIKV. Serological detection of CHIKV is the key to diagnosing and controlling the prevalence of this virus. In this study, we review the progress of the serological detection of the envelope (E) protein in CHIKV, and we provide a novel research assay and ideas for the serological detection of CHIKV. The luciferase immunosorbent assay (LISA) does not require species-specific labeled secondary antibodies for detection, making it universally suitable for tracking samples from various animals or carriers. At present, most research on CHIKV antigen detection technology tends to combine two or more proteins to avoid the decrease in detection ability caused by antigen mutation. Our results indicate that two or more kinds of CHIKV E antigens combined with LISA detection can improve the detection rate of anti-CHIKV immunoglobulin G (IgG) antibodies in CHIKV-infected patient sera and detect antibodies in the early stage of infection accurately and sensitively. After 235 days of infection, the anti-CHIKV IgG antibodies could still be detected in CHIKV-infected patients. All serum samples were tested with a detection rate of 100% after combining various recombinant CHIKV E antigens. Our proposed CHIKV-specific LISA could be a useful tool for serum diagnosis of CHIKV infection and serum epidemic research in areas where CHIKV is endemic, which would help to manage potential epidemics in the future.IMPORTANCE At present, chikungunya virus (CHIKV) is still circulating in some parts of the world, and mutated strains have emerged, making it easier for the virus to spread among humans. With the continuous variation of CHIKV, its antigen variation leads to the decline of detection ability. In addition, the risk of transmission of CHIKV in areas where CHIKV is not endemic, such as China, has increased dramatically, which compels us to enhance the detection capacity of CHIKV and continuously monitor CHIKV antibody levels in the population. Real-time quantitative PCR (RT-PCR) detection technology will not be reliable when the infection time is chronic or in subclinical infection due to decreases in virus concentration, and an antibody detection technology must be adopted. In this study, multiple CHIKV envelope (E) antigens were used to detect anti-CHIKV IgG antibodies in serum for the first time. The new assay is characterized by convenient operation, high detection rate, and high sensitivity and has significance for early warning and monitoring. Moreover, it contributes to the prevention and control of CHIKV.

Comparative evaluation of Panther Fusion and real-time PCR for detection of Burkholderia pseudomallei in spiked human blood.

Introduction. Melioidosis is an infection that most commonly presents with bacteraemia. Culture-based laboratory methods can result in a significant delay to organism identification. Molecular diagnostic techniques have a high sensitivity and rapid time to diagnosis. A decreased time to diagnosis is likely to improve patient outcomes. Aim. To compare the Panther Fusion automated molecular instrument to an in-house method for the detection of Burkholderia pseudomallei directly from spiked human whole-blood samples. Results. The in-house method detected 11/12 (92 %) samples with a B. pseudomallei concentration of 2.5–4.5×102 c.f.u. ml−1. The Panther was less reliable, detecting only 8/14 (75 %) samples with a similar bacterial concentration. The Panther was able to detect 12/12 (100 %) spiked blood culture-positive samples. Conclusion. The direct detection of B. pseudomallei from patient blood on presentation to a healthcare facility will significantly decrease time to diagnosis. We describe an in-house real-time PCR method with the lowest reported limit of detection to date. Due to lower sensitivity, the Panther Fusion would be best used as a diagnostic method directly from a positive blood culture.

A sustainable approach for removing nitrate: Studying the nitrate transformation and metabolic potential under different carbon source by microorganism

Excessive nitrate is a critical concern for a long-term greenhouse production system and potentially pollutes groundwater through leaching. Bacillus megaterium NCT-2 can be used to remove excessive nitrate. Enrichments were made from salinized soil, with and without strain NCT-2, to examine the influence of Bacillus megaterium NCT-2 on the nitrate metabolic potential and microbial composition under various carbon sources using detection of nitrogen content, high-throughput sequencing and quantitative real-time PCR. The results demonstrated that strain NCT-2 enhanced the ability of enriched microorganisms to convert nitrate to ammonium with some but not all carbon sources. The microbial composition and their functional potential had a significant relationship with the strain NCT-2 and carbon sources in the concentrates. Specifically, the enrichments with strain NCT-2 significantly increased the relative abundance of Bacillus and Pseudomonas genera, while decreased the relative abundance of Halomonas genus compared to enrichments without NCT-2 strain. Furthermore, in terms of carbon sources, D-cellobiose promoted the enrichment of Enterobacter genus, methyl-glucoside favored the enrichments of Photobacterium or Serratia genera, and D-mannitol favored the enrichments of Photobacterium, Serratia, or Halomonas genera. In summary, the application of Bacillus megaterium NCT-2 will increase the potential of microorganisms to remove nitrate from the environment. Moreover, the current study demonstrates that different carbon sources can also increase the potential for excess nitrate removal by changing the microbial community composition.

The Oxidative Injury of Extracellular Hemoglobin Is Associated With Reactive Oxygen Species Generation of Grass Carp (Ctenopharyngodon idella).

Intravascular hemolysis is a fundamental feature of hemorrhagic venereal infection or tissue and releases the endogenous damage-associated molecular pattern hemoglobin (Hb) into the plasma or tissues, which results in systemic inflammation, vasomotor dysfunction, thrombophilia, and proliferative vasculopathy. However, how the cytotoxic Hb affects the tissues of grass carp remains unclear. Here, we established a hemolysis model in grass carp by injecting phenylhydrazine (PHZ). The data revealed that the PHZ-induced hemolysis increased the content of Hb and activated the antioxidant system in plasma. The histopathology analysis data showed that the PHZ-induced hemolysis increased the accumulation of Hb and iron both in the head and middle kidney. The results of quantitative real-time PCR (qRT-PCR) detection suggested that the hemolysis upregulated the expressions of iron metabolism-related genes. In addition, the immunofluorescence and immunohistochemistry data revealed that the hemolysis caused an obvious deposition of collagen fiber, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) accumulation and increased the content of oxidative-related enzymes such as β-galactosidase (β-GAL), lipid peroxide (LPO), and MDA in both the head and middle kidney. Furthermore, the PHZ-induced hemolysis significantly increased the production of reactive oxygen species (ROS), which resulted in apoptosis and modulated the expressions of cytokine-related genes. Taken together, excess of Hb released from hemolysis caused tissue oxidative damage, which may be associated with ROS and inflammation generation.

A specific gene, TSA, used as endogenous reference gene for qualitative and real-time quantitative PCR detection of Termitomyces albuminosus

As a nutritious and high-value wild edible mushroom, Termitomyces albuminosus is very popular among people. However, this has caused many unscrupulous businessmen to adulterate it in order to gain huge profits. Qualitative and quantitative PCR identification based on the endogenous reference gene has been widely used to detect the source components of the species. In this work, we screened a large number of DNA sequence of T. albuminosus in the GenBank database by BLAST comparison and interspecies homology comparison, and finally the TSA gene was selected as the candidate. Then, primers were designed for the species-specific regions of the TSA gene, and the specificity was verified by qualitative PCR with 18 other mushroom species. In addition, qualitative and quantitative PCR sensitivity assays were performed, and the limits of detection were 2.56 ng and 200 pg respectively. The detection system was also applied in the deep processed products, the results were consistent with those in the labeled ingredients. In conclusion, we have proposed the TSA gene as an endogenous reference gene for T. albuminosus for the first time, which providing an effective assistance to protect consumers' benefits and promote the positive development of the T. albuminosus industry.

Development of a duplex real-time PCR assay for simultaneous detection and differentiation of Theileria equi and Babesia caballi.

Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.

CREST, a Cas13-Based, Rugged, Equitable, Scalable Testing (CREST) for SARS-CoV-2 Detection in Patient Samples.

The COVID-19 pandemic has taken a devastating human toll worldwide. The development of impactful guidelines and measures for controlling the COVID-19 pandemic requires continuous and widespread testing of suspected cases and their contacts through accurate, accessible, and reliable methods for SARS-CoV-2 detection. Here we describe a CRISPR-Cas13-based method for the detection of SARS-CoV-2. The assay is called CREST (Cas13-based, rugged, equitable, scalable testing), and is specific, sensitive, and highly accessible. As such, CREST may provide a low-cost and dependable alternative for SARS-CoV-2 surveillance. © 2022 Wiley Periodicals LLC. Basic Protocol: Cas13-ased detection of SARS-CoV-2 genetic material using a real-time PCR detection system Alternate Protocol: Cas13-based detection of SARS-CoV-2 genetic material using a fluorescence viewer Support Protocol 1: LwaCas13a purification Support Protocol 2: In vitro transcription of synthetic targets.

SARS-CoV-2 Direct Real Time PCR without RNA Extraction: Appropriate Method or Not?

Real time PCR (RT-PCR) detection method is the widely used for COVID-19 virus detection. This includes sample collection in viral transport medium (VTM), viral RNA extraction followed by detection of virus using fluorescence dye-based system using RT-PCR machine. Several studies have demonstrated a new method which replaces the extraction step by a simple method involving DTT and Proteinase-K and heat treatment. ICMR and few other governing bodies have approved such protocols but are they appropriate in clinical context? In present study, we tried to evaluate one such protocol by using ICMR and WHO approved COVID-19 detection protocol (of CoviPath™ COVID-19 RT-PCR Kit) by replacing RNA extraction step. We used 228 clinical COVID-19 samples for studying method which includes 176 positive (CT values from 14 to 23; 24 to 31 and 32 to 37 were considered as high, moderate and low positive respectively) and 52 negative nasopharyngeal and oropharyngeal swabs samples. We got 100% concordant results with negative samples and 92% concordant and 8% non-concordant results for positive samples. Non-concordant results are with low positive samples. Low level of positivity in the samples could indicate the initial/end stage of COVID-19 disease. If they are at the initial stage, they can be the potential carrier and spread the disease. Authors believe that direct methods can be used for screening bit not for diagnosis of COVID-19 disease.

Viral aetiology in paediatric age group patients admitted with acute febrile encephalopathy in Western Rajasthan

PurposeIn this study, we are trying to find out viral aetiology in paediatric age group patients from 1 month to 15 years of age in Western Rajasthan region. MethodsA total of 105 patients from 1 month to 15 years were recruited into this study. CSF samples were collected and were processed by multiplex real-time PCR for detection of various predefined panels of viral agents. ELISA was also done for all samples for detection of dengue, JE, measles and mumps. ResultsA total of 32 samples out of 105 were tested positive for viral agents. Viral aetiology detected in this study were Adenovirus (n ​= ​2), EBV (n ​= ​1), HHV-1 (n ​= ​10), HHV-6 (N ​= ​5), Parechovirus (n ​= ​1), Parvovirus B19 (n ​= ​7), Dengue (n ​= ​2) and Measles (n ​= ​1). Mixed infections were also detected, HHV-1 and HHV-6 (n ​= ​2), HHV-1 and Parvovirus B19 (n ​= ​1). In 73 patients no viral aetiology could be detected. ConclusionsParvovirus B19 is sporadically prevalent in this geographical region. In this study, HHV-6 was also found which has not been reported earlier from India.

LncRNA TMPO antisense RNA 1 promotes the malignancy of cholangiocarcinoma cells by regulating let-7g-5p/ high-mobility group A1 axis

ABSTRACT Cholangiocarcinoma (CHOL) is often diagnosed at an advanced stage; therefore, exploring its key regulatory factors is important for earlier diagnosis and treatment. This study aimed to identify the mechanisms of long non-coding RNA (lncRNA) TMPO Antisense RNA 1 (TMPO-AS1), microRNA let-7 g-5p, and high-mobility group A1 (HMGA1) proteins in CHOL. Our results, through quantitative real-time PCR and Western blot detection, showed that TMPO-AS1 and HMGA1 were overexpressed while let-7 g-5p was underexpressed in CHOL. Cell function experiments in CHOL cells revealed that TMPO-AS1 knockdown inhibited cell proliferation, colony formation, and cell migration, but induced apoptosis. TMPO-AS1 knockdown also suppressed tumor growth in vivo. Together with luciferase assay and Western blotting, we found that TMPO-AS1 could sponge let-7 g-5p to promote HMGA1 expression. Moreover, HMGA1 overexpression attenuated the effect of TMPO-AS1 downregulation in CHOL cells. Overall, our findings identified the oncogenic effect of TMPO-AS1 on CHOL cells, which may put forward a novel methodology for CHOL diagnosis and therapy.