scholarly journals From primer design to validation of results - is it possible by using free software only? / De la proiectarea primerilor la validarea rezultatelor - este posibil utilizând doar programe gratuite?

2015 ◽  
Vol 23 (2) ◽  
Author(s):  
Adrian Man ◽  
Claudia Bănescu ◽  
Minodora Dobreanu ◽  
Cornel Fraefel
Keyword(s):  
Nucleic Acid ◽  
Molecular Biology ◽  
Molecular Cloning ◽  
Primer Design ◽  
Free Software ◽  
Good Knowledge ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Validation Of Results

AbstractSuccessful experiments in molecular biology require good knowledge about various methods and protocols. In molecular biology, nucleic acid manipulation is the essence, starting with the quality of extraction and ending with several analysis assays (PCR, RT-PCR, qPCR, PCR arrays, molecular cloning, etc). Though many of these are so called “standardized”, in practice there are many variables that can influence the outcome of the experiment. Due to the importance of optimal primer design in PCR assays, we will focus on primer designing and checking software, but we also present other useful free tools that can help researchers in the molecular biology field

scholarly journals A COVID-19 patient with multiple negative results for PCR assays outside Wuhan, China: a case report

2020 ◽  
Author(s):  
Li-Da Chen ◽  
Hao Li ◽  
Yu-Ming Ye ◽  
Zhi Wu ◽  
Ya-Ping Huang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Clinical Manifestations ◽  
Vital Role ◽  
Human Interferon ◽  
Nasopharyngeal Swab ◽  
Lung Field ◽  
Rt Pcr ◽  
Negative Results ◽  
Lower Lung ◽  
Pcr Assays

Abstract Background: The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a public health emergency of major international concern. Real-time RT-PCR assays are recommended for diagnosis of COVID-19. Here we report a rare case of COVID-19 with multiple negative results for PCR assays outside Wuhan, China. Case presentation: A 32-year old male was admitted to our hospital because of 6 days of unexplained fever on January 29, 2020. He had come from Wuhan city 10 days before admission. 5 days before admission, no abnormality was noted in laboratory test, chest radiography, and nasopharyngeal swab test for the SARS-CoV-2 nucleic acid. The patient was treated with ibuprofen for alleviating fever. On admission, chest computed tomography showed multiple ground-glass opacities in right lower lung field. COVID-19 was suspected. 3 times of nasopharyngeal swab specimens were collected after admission. However, none of the specimens were positive. The patient was confirmed with COVID-19 after fifth SARS-CoV-2 nucleic acid test. He was treated with lopinavir/ritonavir, recombinant human interferon alfa-2b inhalation, methylprednisolone. After 18 days of treatment, he was discharged with improved symptoms, lung lesions and negative results of nasopharyngeal swab. Conclusion: This case reminds clinician that a patient with high clinical suspicion of COVID-19 but multiple negative RT-PCR result should not be taken out of isolation. A combination of patient’s exposure history, clinical manifestations, laboratory tests, and typical imaging findings plays a vital role in making preliminary diagnosis and guide early isolation and treatment. Repeat swab tests are helpful in diagnosis for this kind of patients.

scholarly journals SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection

2022 ◽  
Author(s):  
Karrie K. K. Ko ◽  
Nurdyana Binte Abdul Rahman ◽  
Shireen Yan Ling Tan ◽  
Kenneth X. L. Chan ◽  
Sui Sin Goh ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Large Scale ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Viral Genomes ◽  
Reverse Transcription Pcr ◽  
Pcr Assays ◽  
Diagnostic Detection ◽  
Complementary Binding

Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples.

Multiplex real-time PCR assays for the prediction of cephalosporin, ciprofloxacin and azithromycin antimicrobial susceptibility of positive Neisseria gonorrhoeae nucleic acid amplification test samples

2020 ◽  
Vol 75 (12) ◽  
pp. 3485-3490 ◽  
Author(s):  
S W Peterson ◽  
I Martin ◽  
W Demczuk ◽  
N Barairo ◽  
P Naidu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
Nucleic Acid Amplification ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Snp Analysis ◽  
Rt Pcr ◽  
Pcr Assays

Abstract Background The incidence of antimicrobial-resistant Neisseria gonorrhoeae (GC) is rising in Canada; however, antimicrobial resistance (AMR) surveillance data are unavailable for infections diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs), representing over 80% of diagnoses. We developed a set of 10 improved molecular assays for surveillance of GC-AMR and prediction of susceptibilities in NAAT specimens. Methods Multiplex real-time PCR (RT–PCR) assays were developed to detect SNPs associated with cephalosporin (ponA, porB, mtrR −35delA, penA A311V, penA A501, N513Y, G545S), ciprofloxacin (gyrA S91, parC D86/S87/S88) and azithromycin [23S (A2059G, C2611T), mtrR meningitidis-like promoter] resistance. The assays were validated on 127 gonococcal isolates, 51 non-gonococcal isolates and 50 NAATs with matched culture isolates. SNPs determined from the assay were compared with SNPs determined from in silico analysis of WGS data. MICs were determined for culture isolates using the agar dilution method. Results SNP analysis of the 50 NAAT specimens had 96% agreement with the matched culture RT–PCR analysis. When compared with MICs, presence of penA A311V or penA A501 and two or more other SNPs correlated with decreased susceptibility and presence of three or more other SNPs correlated with intermediate susceptibility to cephalosporins; presence of any associated SNP correlated with ciprofloxacin or azithromycin resistance. NAAT-AMR predictions correlated with matched-culture cephalosporin, ciprofloxacin and azithromycin MICs at 94%, 100% and 98%, respectively. Conclusions We expanded molecular tests for N. gonorrhoeae AMR prediction by adding new loci and multiplexing reactions to improve surveillance where culture isolates are unavailable.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Ute Eberle ◽  
◽  
Clara Wimmer ◽  
Ingrid Huber ◽  
Antonie Neubauer-Juric ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Pcr Assays ◽  
Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

scholarly journals Prolonged SARS-CoV-2-RNA Detection from Nasopharyngeal Swabs in an Oncologic Patient: What Impact on Cancer Treatment?

2021 ◽  
Vol 28 (1) ◽  
pp. 847-852
Author(s):  
Anna Ferrari ◽  
Marco Trevenzoli ◽  
Lolita Sasset ◽  
Elisabetta Di Liso ◽  
Toni Tavian ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Treatment ◽  
Cancer Patients ◽  
Clinical Symptoms ◽  
Palliative Radiotherapy ◽  
Rt Pcr ◽  
Cancer Treatments ◽  
Global Challenge ◽  
Viral Culture ◽  
Pcr Assays

The pandemic of SARS-CoV-2 is a serious global challenge affecting millions of people worldwide. Cancer patients are at risk for infection exposure and serious complications. A prompt diagnosis of SARS-CoV-2 infection is crucial for the timely adoption of isolation measures and the appropriate management of cancer treatments. In lung cancer patients the symptoms of infection 19 may resemble those exhibited by the underlying oncologic condition, possibly leading to diagnostic overlap and delays. Moreover, cancer patients might display a prolonged positivity of nasopharyngeal RT-PCR assays for SARS-CoV-2, causing long interruptions or delay of cancer treatments. However, the association between the positivity of RT-PCR assays and the patient’s infectivity remains uncertain. We describe the case of a patient with non-small cell lung cancer, and a severe ab extrinseco compression of the trachea, whose palliative radiotherapy was delayed because of the prolonged positivity of nasopharyngeal swabs for SARS-CoV-2. The patient did not show clinical symptoms suggestive of active infection, but the persistent positivity of RT-PCR assays imposed the continuation of isolation measures and the delay of radiotherapy for over two months. Finally, the negative result of SARS-CoV-2 viral culture allowed us to verify the absence of viral activity and to rule out the infectivity of the patient, who could finally continue her cancer treatment.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals Combined SARS-CoV-2 nucleic acid amplification testing and respiratory virus panel RT-PCR on the Hologic Panther Fusion system

2021 ◽  
Vol 138 ◽  
pp. 104792
Author(s):  
Bryan A. Stevens ◽  
Catherine A. Hogan ◽  
Kenji O. Mfuh ◽  
Ghazala Khan ◽  
Malaya K. Sahoo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Respiratory Virus ◽  
Nucleic Acid Amplification ◽  
Fusion System ◽  
Rt Pcr ◽  
Nucleic Acid Amplification Testing

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

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