scholarly journals Verification and diagnostic evaluation of the RealStar® Middle East respiratory syndrome coronavirus (N gene) reverse transcription-PCR kit 1.0

2019 ◽  
Vol 14 (11) ◽  
pp. 941-948 ◽  
Author(s):  
Leonie-Sophie Hecht ◽  
Angeles Jurado-Jimenez ◽  
Markus Hess ◽  
Hussein El Halas ◽  
Gregor Bochenek ◽  
...  

Aim: We report the diagnostic evaluation of a confirmatory reverse transcription-PCR (RT-PCR) kit targeting the Middle East respiratory syndrome coronavirus (MERS-CoV) N gene. Material & methods: 33 patient samples from two collections sites in Riyadh, Saudi Arabia, which were pre-characterized via real-time RT-PCR targeting MERS-CoV orf1a and upE, and were tested using the MERS-CoV N gene, as a confirmatory assay. This diagnostic procedure follows a two-step diagnostics scheme, recommended by the WHO. Results: 18/33 samples tested positive, 11/33 tested negative for MERS-CoV RNA and 2/33 showed uncertain results. Conclusion: The results suggest, that the RealStar® MERS-CoV (N gene) RT-PCR kit 1.0 can be considered a suitable and reliable confirmatory assay in combination with the RealStar MERS-CoV RT-PCR kit 1.0 according to the diagnostic scheme recommended by WHO.

1998 ◽  
Vol 36 (12) ◽  
pp. 3463-3467 ◽  
Author(s):  
H. Vanderhallen ◽  
F. Koenen

The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3′ end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.


2013 ◽  
Vol 52 (1) ◽  
pp. 67-75 ◽  
Author(s):  
X. Lu ◽  
B. Whitaker ◽  
S. K. K. Sakthivel ◽  
S. Kamili ◽  
L. E. Rose ◽  
...  

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4903-4903 ◽  
Author(s):  
Ibraheem H Motabi ◽  
Syed Ziauddin A. Zaidi ◽  
Mamoun Hassan Ibrahim ◽  
Imran K Tailor ◽  
Nawal Faiez Alshehry ◽  
...  

Abstract Introduction Respiratory viruses are an important cause of outbreaks of pneumonia in hematological malignancy patients. Recently, novel Middle East respiratory syndrome coronavirus (MERS-CoV) caused a cluster of life-threatening infections in Saudi Arabia (688 confirmed MERS-CoV infection cases with 282 deaths were reported to WHO by June 6, 2014 including 28% cases in HCW). Most patients had upper &/or lower respiratory tract symptoms but other features included abdominal pain, diarrhea, acute kidney injury & shock. Few hematology units were closed due to havoc. Here we report clinical features & outcome of 4 patients diagnosed at our unit during the peak period (mid-March through May 2015) including 2 who got chemotherapy (chemo) soon after recovery. Patient #1 A 62-yr-old male had free λ light chain multiple myeloma (MM) with spinal cord compression. After surgery & local radiation, he was sent to us with paraplegia & grade 4 infected sacral bedsore that needed inpatient care. He was started on CyBorD Cycle 2 on April 24, 2014. On May 2, he had a fever spike with shortness of breath (SOB) & cough. He was started on antibiotics. CXR revealed bilateral infiltrates & right sided pleural effusion. His O2 sat dropped & he needed CPAP. Oseltamivir was started & sputum was positive for MERS-CoV RT-PCR. He became afebrile with decreasing O2 requirement, CXR normalized & RT-PCR for MERS-CoV turned negative. Later he was able to receive 3rdcycle of CyBorD. Patient #2 A 65-yr-old lady came to us with B symptoms & huge organomegaly due to stage-IV DLBCL. On May 2, 2014 RCVP chemo was started. On day 6, she spiked fever with SOB, cough & was started on imipenem. CXR showed consolidation in right lower lobe. She needed 4L of O2/min. On May 10, 2014, she worsened with RR 32/min, O2 sat 79% on 15L O2/min & BP 79/47 mmHg. CXR revealed bilateral consolidation. She needed intubation & inotropic support in ICU. Vancomycin & oseltamivir were started & RT-PCR was positive for MERS-CoV on two nasopharyngeal swabs (NPS). LFT & RFT were normal but she continued to decline & died on May 13, 2014. Patient # 3 A 22-yr-old lady with past H/O AML t(8;21) was admitted on April 27, 2014 with 3 day H/O cough, fever & SOB. CXR had infiltrates in left lower lobe. She had severe pancytopenia & BMB confirmed relapsed AML. She had slightly raised LFT. Urine grew Ent. fecium. Antibiotics & voriconazole were used. She remained febrile over next 2 days. CT chest revealed extensive bilateral consolidation. She needed O2 up to 5L/Min for few days. RT-PCR for MERS-CoV was positive from NPS. She was initiated on oseltamivir. She became afebrile after 2 days & repeated RT-PCR for MERS-CoV was negative. Fludara, Ara-C (FA) chemo was started. She remained neutropenic for next 4 weeks but there was no recurrence of respiratory symptoms. BMB on day 28 of FA confirmed CR. CT chest revealed complete resolution of air space opacities. She was discharged with plan to undergo matched sibling donor Allo-HSCT. Patient #4 A 76-yr-old male with H/O HTN & CKD was diagnosed to have IgA κ MM. He was started on MPV chemo as inpatient due to logistic reasons. After 3 cycles of MPV, serum free κ chains decreased by 91% but remained on dialysis. On 21 April, 2014 he developed cough, SOB & fever. CXR revealed bilateral infiltrates & antibiotics were started. He worsened over next few days & CXR showed worsening bilateral consolidation. Eventually he needed intubation. He was treated with antimicrobials including voriconazole & oseltamir. RT-PCR for MERS-CoV was positive from NPS. Unfortunately he died few days later. Discussion: Patients with hematological malignancies are at increased risk of community & hospital-acquired infections. Recent outbreak of MERS-CoV infection has created a havoc among hematologists community. There is uncertainty about impact of MERS-CoV infection on continuation of chemo. We report 4 cases of hematological malignancies with MERS-CoV infection. Three of the 4 patients developed severe pneumonia & required intubation (2 died later) & one had milder form of pneumonia treated in isolation room. In addition to supportive care, all 4 received antimicrobials & oseltamivir. Chemo was safe soon after recovery from infection in the surviving 2 patients. We propose that during MERS-CoV epidemics, pneumonia can be treated with supportive care, antibiotics & oseltamivir. Chemo can be continued for the malignant disease soon after recovery. Further reports are needed to confirm our findings. Disclosures No relevant conflicts of interest to declare.


Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.


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