Sensitive Polymerase Chain Reaction Research Articles

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Loop-Mediated Isothermal Amplification Assay Using Gold Nanoparticles for Detecting Treponema pallidum subspp. pallidum.

Venereal syphilis in humans is caused by Trepenoma pallidum subspp. pallidum. A study has shown that 30,302 individuals in Thailand had syphilis in 2020, with a male-to-female ratio of 1:0.8 and the highest incidence rate at ages between fifteen and twenty-four. This research aimed to develop a loop-mediated isothermal amplification assay using gold nanoparticles (LAMP-AuNPs). Analytical sensitivity, diagnostic specificity, accuracy, and predictive values for each technique are provided. The diagnosis sensitivities of polymerase chain reaction using agarose gel electrophoresis (PCR-AGE), loop-mediated isothermal amplification assay using agarose gel electrophoresis (LAMP-AGE), and LAMP-AuNPs were 116 ng/µL, 11.6 ng/µL, and 11.6 ng/µL, respectively. We evaluated the analytical specificity using PCR and a LAMP-based assay, and there was no cross-reactivity to Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy humans. After analyzing 400 serum samples of patients suspected of syphilis, the LAMP-AGE and LAMP-AuNPs assays displayed 100% diagnostic sensitivity scores, 91% diagnostic specificity scores, 95.5% accuracy rates, 100% positive predictive values (PPVs), and 91% negative predictive values (NPVs), the positive likelihood ratio (LR+) was 11.11, while the negative likelihood ratio (LR-) was 0. Conversely, for PCR assays displayed 100% diagnostic sensitivity scores, 94.5% diagnostic specificity scores, 97.25% accuracy rates, 100% PPVs, and 94.5% NPVs, LR+ was 18.18, and LR- was 0. The LAMP-AuNPs technique demonstrates rapidity, affordability, and convenience, rendering it well-suited for point-of-care applications in the diagnosis, prevention, and management of pathogenic infections.

Diagnosis of invasive aspergillosis in haemato-oncology patients in a routine diagnostic setting.

Invasive Aspergillosis (IA) is a potentially lethal infection in high-risk haemato-oncology patients. Since traditional diagnostic methods have many inherent challenges, Polymerase Chain Reaction (PCR) has been used to diagnose IA. This prospective study evaluated a commercial AsperGenius multiplex real-time PCR for its clinical utility in diagnosing IA compared with galactomannan (GM) testing serum samples from haemato-oncology patients with clinically suspected IA. A total of 107 patients were recruited between April 2022 and March 2023. Serum samples (n=113) collected from those patients for the routine diagnosis by GM Enzyme Linked Immuno-Sorbent Assay (ELISA) were subjected to PCR. The patients were categorised into probable, possible, and no IA based on revised (2020) and previous (2008) European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC-MSG) criteria. The performance characteristics of PCR and GM were calculated against the EORTC criteria by combining probable and possible cases as diseased groups. Among the 107 recruited patients, 93 were categorised into probable/possible IA (diseased group) and 14 into no IA group. The PCR was positive in 53 samples from 49 patients. The sensitivity and specificity of single positive PCR and GM were 51.61% [95% confidence interval, 41-62], 92.86% (66.1-99.8) and 26.88% (18.2-37.1), 92.86% (66.1-99.8), respectively. The combination-based strategy (GM and/or PCR positive) exhibited a moderate sensitivity of 62.37% (51-72.2) and a specificity of 85.71% (57.2-98.2). To conclude, the combined strategy of serum GM and/or PCR positivity, along with radiological findings that fulfilled the EORTC/MSG criteria, has improved the diagnosis of probable IA among high-risk haematological patients with clinically suspected IA.

Diagnosis of bacteria isolated from patients with cutaneous eczema by PCR technique at Azadi Hospital, Kirkuk, Iraq

Eczema is a common skin condition that can be worsened or caused by a bacterial infection. This study aimed to investigate the bacterial pathogens associated with skin eczema in patients admitted to Azadi Hospital in Kirkuk using polymerase chain reaction (PCR) technology. The goal was to enhance the accuracy and efficiency of bacteria identification compared to traditional methods. A total of [300] patients suffering from symptoms of skin eczema between the ages of 1-40 years, females and males, were included. Bacterial isolates were obtained from skin samples, and conventional microbiological techniques were initially used for identification. Subsequently, molecular analysis using PCR was performed to confirm and characterize the bacterial species. The study included 165 male patients represented (55%), 135 female (45%), of participants who had symptoms of skin eczema. The PCR test targeted specific genes associated with common bacteria implicated in skin infections, such as Staphylococcus. aureus (33 %), Staphylococcus epidermidis (28%), Klebsiella pneumoniae (20 %) , pseudomonas aeruginosa (13 %), respectively, and the remaining( 6 %). of the samples were natural flora. The genetic fingerprints of these bacterial species were identified, an image of them was taken on an agarose gel electrophoreses , and the results were compared with those obtained through traditional methods to evaluate the sensitivity and specificity of PCR in diagnosing bacterial infections in cases of skin eczema. Preliminary results indicated that PCR significantly improved the accuracy and speed of bacterial identification compared to traditional methods. It has allowed the detection of specific bacterial species that might be missed using conventional methods. The prevalence and diversity of bacterial species causing skin eczema in Azadi Hospital in Kirkuk were also explored. This study contributes valuable insights into the microbial etiology of cutaneous eczema, emphasizing the importance of molecular techniques in accurate and rapid diagnosis. The findings may guide more targeted and effective treatment strategies for patients with bacteria-associated skin eczema, ultimately improving patient outcomes and reducing the risk of complications.

Comparison of Polymerase Chain Reaction Method with Culture Method in Antenatal Group B Streptococcus Screening.

The aim of this study was to investigate the prevalence of Group B Streptococcus (GBS) colonization in pregnancies between 35 and 37 weeks of gestation and to compare the effectiveness of polymerase chain reaction (PCR) method with gold standard technique of culture in antenatal GBS screening. Vaginal and rectal swabs of a total of 106 pregnant women between 35th and 37th weeks of gestation, who were admitted to our clinic between January 2022 and August 2022, were evaluated using culture and PCR method. The prevalence of GBS was estimated. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the PCR method were analyzed. The prevalence of GBS was 10.4% and 21.69% using the culture and PCR method, respectively. Compared to the culture, the sensitivity, specificity, PPV, NPV and accuracy of PCR were found to be 100%, 87%, 47%, 100%, and 88%, respectively. This study results suggest that the PCR method is a simple, effective and fast method with high sensitivity, specificity, PPV, and NPV in antenatal GBS screening.

The detection of Salmonella in food based on PCR combined with Pyrococcus furiosus Argonaute

Salmonella is a common zoonotic foodborne bacterial pathogen. In poultry farms, Salmonella can cause pullorum disease, which characterizes decreased appetite and diarrhea in chicks, accompanied by a high mortality rate in the first week of life itself. In pig farms, Salmonella mainly infects piglets and causes porcine paratyphoid, characterizing septicemia and necrotic enteritis and sometimes leading to severe symptoms such as encephalitis. This bacterium poses significant challenges to global public health and food safety. PfAgo (Pyrococcus furiosus Argonaute) is an Argonaute enzyme from the Pyrococcus furiosus, which is utilized in highly sensitive and accurate nucleic acid detection and analysis methods based on the polymerase chain reaction (PCR) combined with PfAgo. In this study, targeting the hilA gene, and can detect as low as 3 × 100 copies and 2.7 × 10°CFU (cells) with no cross-reactivity with other pathogens, it enhances the sensitivity of PCR. The PCR-PfAgo method was successfully applied to detect in clinical food samples including chicken and milk, and the results were consistent with those of traditional bacterial culture. The findings suggest that the PCR-PfAgo method has the potential to replace traditional detection methods and has broad application prospects in the detection of Salmonella pathogen.

A detailed analysis of 16S rRNA gene sequencing and conventional PCR-based testing for the diagnosis of bacterial pathogens and discovery of novel bacteria.

This study represents the first analysis of the bacterial community in chickens affected by swollen head syndrome, utilizing 16S rRNA gene sequencing. Samples were obtained from clinical laying chickens and were examined for the presence of Avibacterium paragallinarum (APG) and Ornithobacterium rhinotracheale (ORT) using conventional polymerase chain reaction (PCR). From the samples, five APG-positive (APG) and APG-negative (N-APG) samples were chosen, along with five specific pathogen-free chickens, for 16S rRNA gene sequencing. Results showed that APG and ORT were widely detected in the chicken samples with swollen head syndrome (SHS, 9/10), while APG was detected in all five specific pathogen-free (SPF) samples. In contrast, conventional PCR sensitivity was found to be inadequate for diagnosis, with only 35.7% (5/14) and 11.1% (1/9) sensitivity for APG and ORT, respectively, based on 16S rRNA gene sequencing data. Furthermore, 16S rRNA gene sequencing was able to quantify the bacteria in the samples, revealing that the relative abundance of APG in the APG group ranged from 2.7 to 81.3%, while the relative abundance of APG in the N-APG group ranged from 0.1 to 21.0%. Notably, a low level of APG was also detected in all 5 SPF samples. The study also identified a significant number of animal and human common bacterial pathogens, including but not limited to Gallibacterium anatis, Riemerella columbina, Enterococcus cecorum, Mycoplasma synoviae, Helicobacter hepaticus, and Staphylococcus lentus. In conclusion, 16S rRNA gene sequencing is a valuable tool for bacterial pathogen diagnosis and the discovery of novel bacterial pathogens, while conventional PCR is not reliable for diagnosis.

Rapid discrimination of African swine fever virus nucleic acid and virions using BenzoNuclease

African swine fever (ASF) is an acute and severe infectious disease caused by the African Swine Fever Virus (ASFV). ASFV exhibits significant resistance and stability in the environment, which, coupled with its double-stranded DNA and large genome, predisposes it to contaminate laboratory samples. This characteristic can lead to false-positive results in swine farm settings even days after disinfection, as detectable through polymerase chain reaction (PCR) or real-time fluorescent quantitative PCR (qPCR) assays. To meet the demand for efficient clinical methods capable of discriminating between ASFV nucleic acid and ASFV virions, this study aims to ascertain the efficacy of the nuclease “BenzoNuclease” in distinguishing ASFV nucleic acid (ASFV-DNA) from ASFV virions. BenzoNuclease is a versatile nucleic acid enzyme with the capacity to degrade nearly all forms of DNA and RNA. Initially, this research established a highly sensitive general PCR detection method for ASFV. Subsequently, a positive control was constructed using the M13 bacteriophage to substitute for active ASFV, facilitating the development of an improved qPCR method. It is important to note that common disinfectants have the potential to deactivate BenzoNuclease. However, in an environment simulating actual production applications, residual disinfectants do not interfere with the enzymatic efficacy of BenzoNuclease, thus not affecting the detection capabilities of this method. Positive clinical samples from pig farms, upon testing with the improved method, revealed that three samples were positive, indicating the presence of viral particles, while the remaining samples were negative, indicating the presence of nucleic acids. This provides an additional new option for sample testing in pig farms.

A multiplex one-step fluorescence quantitative differential diagnosis method for severe hand, foot and mouth disease caused by coxsackievirus A16

Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5’UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/μl, for the 5′ UTR was 50.1187 copies/μl and for the RNaseP gene was 158.49 copies/μl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100 % (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94 % (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95 % (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5 hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.

Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis.

This meta-analysis examines the comparative diagnostic performance of polymerase chain reaction (PCR) for the diagnosis of Pneumocystis pneumonia (PCP) on different respiratory tract samples, in both human immunodeficiency virus (HIV) and non-HIV populations. A total of 55 articles met inclusion criteria, including 11 434 PCR assays on respiratory specimens from 7835 patients at risk of PCP. QUADAS-2 tool indicated low risk of bias across all studies. Using a bivariate and random-effects meta-regression analysis, the diagnostic performance of PCR against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group definition of proven PCP was examined. Quantitative PCR (qPCR) on bronchoalveolar lavage fluid provided the highest pooled sensitivity of 98.7% (95% confidence interval [CI], 96.8%-99.5%), adequate specificity of 89.3% (95% CI, 84.4%-92.7%), negative likelihood ratio (LR-) of 0.014, and positive likelihood ratio (LR+) of 9.19. qPCR on induced sputum provided similarly high sensitivity of 99.0% (95% CI, 94.4%-99.3%) but a reduced specificity of 81.5% (95% CI, 72.1%-88.3%), LR- of 0.024, and LR+ of 5.30. qPCR on upper respiratory tract samples provided lower sensitivity of 89.2% (95% CI, 71.0%-96.5%), high specificity of 90.5% (95% CI, 80.9%-95.5%), LR- of 0.120, and LR+ of 9.34. There was no significant difference in sensitivity and specificity of PCR according to HIV status of patients. On deeper respiratory tract specimens, PCR negativity can be used to confidently exclude PCP, but PCR positivity will likely require clinical interpretation to distinguish between colonization and active infection, partially dependent on the strength of the PCR signal (indicative of fungal burden), the specimen type, and patient population tested.

Precision diagnostics in paediatric dermatology: Advancing management of tinea capitis through dermatophyte PCR.

Tinea capitis (TC) is the most frequent dermatophyte infection in children requiring systemic and topical treatment for several weeks. Traditionally, diagnosis and treatment monitoring were based on microscopic examination and fungal culture of scales and plucked hairs, which both have significant limitations. To investigate the role of dermatophyte polymerase chain reaction (PCR) in the treatment of TC. Scales and plucked hairs of children with TC were investigated by dermatophyte PCR, microscopic examination and fungal culture at baseline and during antifungal treatment. Seventeen children with TC were included. At baseline, sensitivity of PCR was 100% as compared to 60% and 87% for direct microscopy and fungal culture, respectively. Species identification by PCR and fungal culture was consistent in all cases. During follow-up, analysis of 38 samples under treatment showed a sensitivity of PCR, direct microscopy and fungal culture of 68%, 26% and 89% while specificity was 84%, 100% and 100%, respectively. PCR during therapy proved to be false-negative in six and false-positive in three instances. The latter turned negative after 4 weeks without further systemic treatment. Dermatophyte PCR is an excellent tool for baseline diagnostics of TC providing rapid and accurate results. Our findings suggest that due to the fast and reliable results, it may replace direct microscopy and fungal culture to confirm or exclude TC in children. In the treatment course, diagnostic accuracy and performance of PCR seem reduced as compared to fungal culture, limiting its value for treatment monitoring. Mycological cure ascertained by fungal culture should currently remain the therapeutic goal.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis.

Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

Origin of pathogenic variant and mosaicism in families with a sporadic case of haemophilia B.

Of newly diagnosed cases of haemophilia B, the proportion of sporadic cases is usually 50% of severe cases and 25% of moderate/mild cases. However, cases presumed to be sporadic due to family history may not always be sporadic. Few case reports have been published on mosaicism in haemophilia B. The present study aimed to trace the origin of the pathogenic variant in a well-defined cohort of sporadic cases of haemophilia B by haplotyping markers. It also aimed to determine the frequency of mosaicism in presumed non-carrier mothers. The study group was 40 families, each with a sporadic case of haemophilia B analysed in two-to-three generations by Sanger sequencing, haplotyping and using the sensitive droplet digital polymerase chain reaction (ddPCR) technique. In 31/40 (78%) of the families, the mother carried the same pathogenic variant as her son, while Sanger sequencing showed that 9/40 (22%) of the mothers did not carry this variant. Of these variants, 2/9 (22%) were shown to be mosaics by using the ddPCR technique. 16/21 carrier mothers, with samples from three generations available, had a de novo pathogenic variant of which 14 derived from the healthy maternal grandfather. The origin of the pathogenic variant in sporadic cases of haemophilia B is most often found in the X-chromosome derived from the maternal grandfather or, less often, from the maternal grandmother. Mosaic females seem to be found at the same frequency as in haemophilia A but at a lower percentage of the pathogenic variant.

Diagnostic Approaches to Helicobacter pylori: A Comparative Study of Detection in Gastric Biopsy and Aspirates.

Background Helicobacter pyloriis one of the most common bacterial pathogens in humans. It is a microaerophilic bacteria with multiple unipolar flagella. It is associated with the development of various lesions like chronic gastritis, gastric ulcers, adenocarcinoma, and mucosa-associated lymphomas. The aim of this study was a comparative evaluation of the rapid urease test (RUT) and polymerase chain reaction (PCR) in gastric biopsy and aspirates for the detection ofH. pyloriinfection and to further determine the sensitivity and specificity of RUT and PCR. Method Endoscopic guided biopsy tissue and gastric aspirate specimens were collected from 110 patients with symptoms like gastritis, dyspepsia, etc., and subjected to RUT and PCR for detection ofH. pyloriinfection. Results A total of 110 samples, including both biopsy tissue (77) and gastric aspirate (33) were subjected to RUT and PCR. RUT for biopsy tissue showed the highest sensitivity (97.18%), compared to gastric aspirate (78.94%). Comparing RUT with PCR, the sensitivity and specificity of PCR were 93.33% and 90.0%, respectively. The positive predictive value (PPV) of PCR was 97.67%, the negative predictive value (NPV) was 75.0%, and the accuracy was 92.73%. Conclusion The present study showed that RUT is a rapid and accurate invasive test for the detection ofHelicobacter pyloriinfection in biopsy tissue as compared to gastric aspirate specimens, which are more sensitive to PCR. The study also showed that biopsy tissue was found to be a superior specimen for the detection ofHelicobacter pylorias compared to gastric aspirate.

Detection of Clostridioides difficile infection by assessment of exhaled breath volatile organic compounds

Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired infective diarrhea. Current methods for diagnosing CDI have limitations; enzyme immunoassays for toxin have low sensitivity and Clostridioides difficile polymerase chain reaction cannot differentiate infection from colonization. An ideal diagnostic test that incorporates microbial factors, host factors, and host-microbe interaction might characterize true infection. Assessing volatile organic compounds (VOCs) in exhaled breath may be a useful test for identifying CDI. To identify a wide selection of VOCs in exhaled breath, we used thermal desorption-gas chromatography-mass spectrometry to study breath samples from 17 patients with CDI. Age- and sex-matched patients with diarrhea and negative C.difficile testing (no CDI) were used as controls. Of the 65 VOCs tested, 9 were used to build a quadratic discriminant model that showed a final cross-validated accuracy of 74%, a sensitivity of 71%, a specificity of 76%, and a receiver operating characteristic area under the curve of 0.72. If these findings are proven by larger studies, breath VOC analysis may be a helpful adjunctive diagnostic test for CDI.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Improving polymerase chain reaction diagnostic rates for herpes simplex keratitis: results of a pilot study.

Laboratory confirmation is crucial for diagnosis and management of herpes simplex virus (HSV) keratitis. However, the sensitivity of polymerase chain reaction (PCR) in keratitis is low (25%) compared with that of mucocutaneous disease (75%). We developed an educational intervention aimed at improving the diagnostic yield of PCR. The medical records of keratitis cases seen at the emergency department of a London tertiary ophthalmic referral hospital over two distinct periods, before and after an educational program on swab technique, were reviewed retrospectively. A total of 252 HSV cases were included. Increases in the laboratory-confirmed diagnosis of HSV-1 were observed, in both first presentations (11.1%-57.7%) and recurrent cases (20%-57.6%). The rate of positive HSV-1 PCR in eyes with an epithelial defect increased from 19% pre-intervention to 62% post intervention. Notably, 3% were positive for varicella zoster virus DNA, and there was a single case of Acanthamoeba keratitis. Our results suggest that, with proper swabbing technique, PCR may be more sensitive than previously reported.

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Polymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer-template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer-template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different types of DNA polymerases were used. Notably, when a single-nucleotide mismatch occurred at the 3' end of the primer, we observed significant decreases in the analytical sensitivity (0-4%) with Invitrogen™ Platinum™ Taq DNA Polymerase High Fidelity, while the analytical sensitivity remained unchanged with Takara Ex Taq Hot Start Version DNA Polymerase. Leveraging these findings, we designed a highly specific PCR to amplify Babesia while effectively avoiding the genetically close Theileria. Through elucidating the critical interplay between types of DNA polymerases and primer-template mismatches, this research provides valuable insights for improving PCR accuracy and performance. These findings have important implications for researchers aiming to achieve robust qPCR results in various molecular biology applications.

CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA.

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.