ABSTRACT
The outbreak of COVID-19 makes epidemic prevention and control become a growing global concern. Nucleic acid amplification testing (NAAT) can realize early and rapid detection of targets, thus it is considered as an ideal approach for detecting pathogens of severe acute infectious diseases. Rapid acquisition of high-quality target nucleic acid is the prerequisite to ensure the efficiency and accuracy of NAAT. Herein, we proposed a simple system in which magnetic nanoparticles (MNPs) based nucleic acid extraction was carried out in a plastic Pasteur pipette. Different from traditional approaches, this proposed system could be finished in 15 min without the supports of any electrical instruments. Furthermore, this system was superior to traditional MNPs based extraction methods in the aspects of rapid extraction and enhancing the sensitivity of a NAAT method, accelerated denaturation bubbles mediated strand exchange amplification (ASEA), to the pathogens from various artificial samples. Finally, this Pasteur pipette system was utilized for pathogen detection in actual samples of throat swabs, cervical swabs and gastric mucosa, the diagnosis results of which were identical with that provided by hospital. This rapid, easy-performing and efficiency extraction method ensures the applications of the NAAT in pathogen detection in regions with restricted resources.
Subject(s)
Infections/diagnosis , Magnetite Nanoparticles , Nucleic Acid Amplification Techniques/methods , Nucleic Acids/isolation & purification , COVID-19/diagnosis , Helicobacter Infections/diagnosis , Helicobacter pylori/isolation & purification , Human papillomavirus 16/isolation & purification , Humans , Papillomavirus Infections/diagnosis , Pneumonia, Mycoplasma/diagnosis , SARS-CoV-2/isolation & purificationABSTRACT
OBJECTIVE: To validate the colposcopy indication proposed by the 2019 ASCCP Risk-Based Management Consensus Guidelines for abnormal cervical cancer screening tests (the 2019 ASCCP guidelines). METHODS: Clinical data of 1404 patients who underwent colposcopy in single center in China were reviewed. Based on history and current cervical screening (HPV & cytology), corresponding recommendations were given according to the 2019 ASCCP guidelines. The agreement and discrepancy of colposcopy indication were analyzed between the Chinese consensus and the 2019 ASCCP guidelines. RESULTS: Colposcopy indication was matched in about 80% patients. The left 20% were recommended with follow-up by the 2019 ASCCP guidelines. The discrepancy mainly focused on patients having a current result of HPV-positive NILM without unknown history. The ratio of observed CIN3+ in our database over estimated CIN3+ by the 2019 ASCCP guidelines was 6.2 (31/5). The ratio was even higher in patients with HPV16/18-positive NILM (7, 28/4), compared with those with other types of high-risk HPV-positive NILM (3, 3/1). The 2019 ASCCP guidelines had a relatively high sensitivity (83.1%), a low specificity (21.5%), a low positive predictive value (14.1%) and a high negative predictive value (89.1%) for prediction of CIN 3+. CONCLUSIONS: We could try to apply the 2019 ASCCP guidelines in Chinese population. The classification of HR-HPV was strongly recommended during risk assessment. For patients with HPV16/18 infection, colposcopy should be recommended. Perspective multi-center randomized controlled trial with reliable follow-up should be performed in the future to confirm the feasibility.
Subject(s)
Colposcopy/standards , Early Detection of Cancer/methods , Guidelines as Topic , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosis , Adult , China , Female , Human papillomavirus 16/isolation & purification , Human papillomavirus 18/isolation & purification , Humans , Mass Screening/methods , Papillomavirus Infections/pathology , Retrospective Studies , Risk , Uterine Cervical Neoplasms/pathology , Uterine Cervical Dysplasia/pathologyABSTRACT
DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.