Limitations Of Polymerase Chain Reaction Research Articles

The Relation of Polymerase Chain Reaction and Computed Tomography Infiltrations of Suspected Covid-19 Patients in Emergency Department

Aims: In the context of the persistent global challenge posed by COVID-19, effective diagnostic methods are crucial. Considering the limitations of Polymerase Chain Reaction (PCR) in the Emergency Department (ED), alternative confirmation methods for assessing Covid-19 are needed. This study aimed to evaluate the performance of Thorax-Computed Tomography (T-CT), following Radiological Society of North America (RSNA) recommendations, for suspected Covid-19 patients with pulmonary infiltrations. Materials and Methods: From March to August 2020, 324 ED patients with suspected Covid-19 underwent T-CT scans. Blinded radiologists independently assessed T-CT scans based on RSNA guidelines. Reverse-transcriptase polymerase chain reaction (RT-PCR) served as the reference test. Results: Of 324 patients, 35% tested positive via RT-PCR. T-CT categories were typical (35.2%), indeterminate (47.5%), and atypical (11.1%). Using a typical T-CT category threshold resulted in 66% sensitivity, 81% specificity, 65% positive predictive value (PPV), 82% negative predictive value (NPV), and 76% accuracy. Subgroup analysis of repeat RT-PCR tests improved performance: 80% sensitivity, 79% specificity, 76% PPV, 83% NPV, and 79% accuracy. Combining RT-PCR and T-CT in the ED achieved 95.5% sensitivity, 79% PPV, and 86.4% accuracy. Conclusion: Following RSNA guidelines, T-CT exhibits moderate sensitivity and high specificity for detecting Covid-19. In ED settings with suspected cases, T-CT aids in recommending retesting after an initial negative RT-PCR result, facilitating early management and enabling timely isolation measures. The combined use of RT-PCR and T-CT enhances diagnostic performance, emphasizing the potential benefits of integrating these methods.

Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers

Backgrounds: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required. ResultsIn this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 101 CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 103 CFU/g E. coli O157:H7. ConclusionThis study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.

A Thermocycler Using a Chip Resistor Heater and a Glass Microchip for a Portable and Rapid Microchip-Based PCR Device.

This study proposes a rapid and inexpensive thermocycler that enables rapid heating of samples using a thin glass chip and a cheap chip resistor to overcome the on-site diagnostic limitations of polymerase chain reaction (PCR). Microchip PCR devices have emerged to miniaturize conventional PCR systems and reduce operation time and cost. In general, PCR microchips require a thin-film heater fabricated through a semiconductor process, which is a complicated process, resulting in high costs. Therefore, this investigation substituted a general chip resistor for a thin-film heater. The proposed thermocycler consists of a compact glass microchip of 12.5 mm × 12.5 mm × 2 mm that could hold a 2 μL PCR sample and a surface-mounted chip resistor of 6432 size (6.4 mm × 3.2 mm). Improving heat transfer from the chip resistor heater to the PCR reaction chamber in the microchip was accomplished via the design and fabrication of a three-dimensional chip structure using selective laser-induced etching, a rapid prototyping technique that allowed to be embedded. The fabricated PCR microchip was combined with a thermistor temperature sensor, a blower fan, and a microcontroller. The assembled thermocycler could heat the sample at a maximum rate of 28.8 °C/s per second. When compared with a commercially available PCR apparatus running the same PCR protocol, the total PCR operating time with a DNA sample was reduced by about 20%.

Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients

The objective of the present study was to assess the performance and the best indication of the polymerase chain reaction (PCR) for the detection of Toxoplasmosis gondii DNA in cerebrospinal fluid (CSF) from patients with suspected neurotoxoplasmosis. CSF samples were collected from 79 patients for amplification of the T. gondii genome (gene B1) by two PCR techniques (nested and real time). Twenty-seven of the 79 patients were classified as probable cases of neurotoxoplasmosis on the basis of clinical criteria, neuroimaging and therapeutic response. PCR showed high sensitivity (86.6%) when performed in CSF samples which were collected up to the seventh day of specific toxoplasmosis treatment. There was no positive test after 1week of treatment. These results suggest the usefulness of PCR for the diagnosis of cerebral toxoplasmosis, and support the first week as the window for the best performance of toxoplasmosis PCR in CSF.

The limitations of polymerase chain reaction in the setting of possible recurrent tuberculosis: 2 instructional cases

The interpretation of a positive result for Mycobacterium tuberculosis by nucleic acid amplification such as polymerase chain reaction (PCR) can be challenging. We present 2 cases that illustrate the limitations of tuberculosis PCR on respiratory secretions in previously treated patients, even years after the previous disease episode.

DA BIOLOGIA MOLECULAR À MEDICINA: MÉTODOS COMUMENTE UTILIZADOS EM FARMACOGENÉTICA

Nesta revisão, foram discutidos os procedimentos para obtenção, manuseio e armazenamento de amostras biológicas úteis em testes moleculares. Métodos para triagem e detecção direta de mutações e polimorfismos genéticos foram apresentados. Os princípios, estratégias, aplicações e limitações de variações da reação em cadeia pela polimerase (PCR), tais como eletroforese em gel com gradiente de desnaturação (DGGE), polimorfismo de conformação de fita simples (SSCP), polimorfismo de tamanhos de fragmentos de restrição (RFLP), amplificação alelo oligonucleotídeo-específica (PCR-ASO), PCR em tempo real PCR, arranjos de DNA e outras técnicas moleculares, foram comentados.

Quantitative detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR

The limitation of polymerase chain reaction (PCR) in diagnosis of lower respiratory tract infections (LRTIs) caused by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis has been a distinguishing colonization from infection. We assess here the usefulness of real-time quantitative PCR (RQ-PCR) performed on lower respiratory tract samples to overcome this problem. Consecutive respiratory tract samples from patients with and without signs of infection ( n = 203) were subjected to RQ-PCR, targeting the genes pneumolysin ( S. pneumoniae), fumarate reductase ( H. influenzae), and outer membrane protein B ( M . catarrhalis). DNA from positive controls with predefined colony forming units (CFUs) per milliliter were included to allow estimation of CFU per milliliter for the test samples. In parallel, assessment of quantitative cultures from all samples was performed. In the group of patients with LRTI, significant pathogens (≥10 5 CFU/mL) were found in 32/135 samples (23.7%) with culture, in 51/135 (37.7%) with RQ-PCR, and in 59/135 (43.7%) when combining the methods.

Advantage and limitation of polymerase chain reaction in clinical diagnosis Application

聚合酶链反应(PCR)技术自1983年问世以来,因其对基因或特定核酸序列在短时间内具有极大的扩增效率,已广泛应用于感染性疾病、肿瘤、遗传病、寄生虫病、法医学、动植物和考古等诊断和研究领域,并在某些方面呈现出几乎难以替代的优势.PCR用于疾病的临床诊断后,使人们对蛋白分子表型的认识,进一步深入到了遗传物质--核酸分子的探索,也使临床检验诊断学科中的临床分子诊断技术得到了飞速发展.像任何自然界的事物一样,PCR虽然有其无可比拟的优点,但也有其一定的局限性.如果在临床实际应用中,不遵循客观规则,也很容易产生非PCR技术问题所致的错误检验结果。