Application of cell-free transcription and translation system in CRISPR technologies and the associated biosensors / 生物工程学报

Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid in vitro protein expression. The system bypasses routine laboratory processes such as bacterial transformation, clonal screening and cell lysis, which allows more precise and convenient control of reaction substrates, reduces the impact of bacteria on protein production, and provides a high degree of versatility and flexibility. In recent years, TXTL has been widely used as an emerging platform in clusterd regularly interspaced short palindromic repeat (CRISPR) technologies, enabling more rapid and convenient characterization of CRISPR/Cas systems, including screening highly specific gRNAs as well as anti-CRISPR proteins. Furthermore, TXTL-based CRISPR biosensors combined with biological materials and gene circuits are able to detect pathogens through validation of related antibiotics and nucleic acid-based markers, respectively. The reagents can be freeze-dried to improve portability and achieve point-of-care testing with high sensitivity. In addition, combinations of the sensor with programmable circuit elements and other technologies provide a non-biological alternative to whole-cell biosensors, which can improve biosafety and accelerate its application for approval. Here, this review discusses the TXTL-based characterization of CRISPR and their applications in biosensors, to facilitate the development of TXTL-based CRISPR/Cas systems in biosensors.

CRISPR-based molecular diagnostics: a review / 生物工程学报

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.

Isothermal amplification technology based on microfluidic chip / 生物工程学报

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.

Application evaluation of a rapid fluorescence quantitative PCR method for the detection of SARS-CoV-2 / 中华微生物学和免疫学杂志

Objective:To establish and evaluate a rapid nucleic acid detection method for SARS-CoV-2 based on COYOTE ? Flash20 real-time fluorescent quantitative PCR instrument. Methods:A rapid reaction system was constructed by using specific primer and probe sets targeting ORF1ab and N gene of SARS-CoV-2, and the sensitivity and specificity of the system were verified. At the same time, 108 clinical samples of COVID-19 were used to evaluate the application of this method.Results:The detection method did not require nucleic acid extraction, and the manual operation time was only one minute. After the sample was sent to the system, the test could be completed in 30 minutes. The detection limit of this method was 4×10 2 copies/ml. It had no cross-reactivity with other human coronaviruses (including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV) and other respiratory viruses. The evaluation of clinical sample application showed that the total coincidence rate with the conventional RT-qPCR which required nucleic acid extraction was 98.15%. Conclusions:Through the application evaluation of the rapid fluorescent quantitative PCR method of SARS-CoV-2, it was found that the method was simple, fast, specific and sensitive, and it was suitable for real-time and rapid detection needs in varieties of situations.

Comparison of point-of-care testing and laboratory testing of myocardial damage markers in the diagnosis and prognosis of acute coronary syndrome / 中华急诊医学杂志

Objective To compare the point-of-care testing (POCT) and laboratory testing of myocardial damage markers in the diagnosis and prognosis of acute coronary syndrome (ACS).Methods A total of 3467 patients with ACS who were treated in the Emergency Department Beijing Chaoyang Hospital Affiliated to Capital Medical University between January 1,2006 and June 30,2010 were retrospectively reviewed.The patient demographics (age,sex,past medical history and smoking history) and laboratory testing results (myocardial damage markers,D-dimer,NTproBNP,and ejection fraction [EF]) were analyzed.The patients who received POCT or laboratory testing of myocardial damage markers were compared with regard to emergency department stay (i.e.,the time from the emergency visit to interventional or conservative treatment),cardiovascular events during hospitalization (congestive heart failure,ventricular fibrillation,and cerebrovascular disease),and 28-day mortality rate.Results The emergency department stay,incidence of a cardiovascular event,and 28-day mortality in the POCT group were all lower than that in the laboratory testing group (P =0.000).A prolonged emergency department stay result in an increased incidence of 28-day mortality.The higher level of D-dimer and decreased EF prompted an increased incidence of 28-day mortality.Conclusions Compared with conventional laboratory testing,POCT can significantly shorten the length of an emergency department stay for an ACS patient,decrease the incidence of a cardiovascular event,and improve the prognosis.

Evaluation of HUBI-QUANPRO Point-of-Care Testing for Cardiac Markers / 임상검사와정도관리

BACKGROUND: Recently, quantitative point-of-care testing (POCT) for cardiac markers using colloidal gold particles was developed in Korea. We evaluated the analytical performance of the HUBI-QUANPRO (Humasis, Korea) assay in comparison with two other assays. METHODS: We evaluated the analytical precision and linearity of HUBI-QUANPRO creatine kinase (CK)-MB, cardiac troponin I (cTnI), and B-type natriuretic peptides (BNP). HUBI-QUANPRO assay was compared with ADVIA Centaur (Siemens, Germany) and Triage (Biosite Diagnostics, USA) assays by using 100 blood samples. In addition, we evaluated the interference of hemoglobin on the HUBI-QUANPRO assay. RESULTS: The coefficients of variation of HUBI-QUANPRO CK-MB, cTnI, and BNP were 7.5-9.7%, 12.0-17.4%, and 14.7-15.7%, respectively. The linearity ranges of HUBI-QUANPRO CK-MB, cTnI, and BNP were 4.7-27.8 ng/mL, 0.76-6.51 ng/mL, and 76.2-762.2 ng/mL, respectively. The comparison study showed no significant difference among them. When 0.5% hemolysis occurred, remarkable hemoglobin interference was found in the three markers resulting in underestimation of the concentrations. CONCLUSIONS: HUBI-QUANPRO CK-MB and BNP showed good analytical performances compared with the other two assays. Hemoglobin interference was noted in the HUBI-QUANPRO assay, especially more in BNP. Although the linearity range of cTnI was narrow, its agreement rate with ADVIA Centaur was good, thus the HUBI-QUANPRO assay could be useful as a quantitative POCT for cardiac markers in the emergency department.

Evaluation of CareSens(R) N Glucometer Glucose Monitoring System / 임상검사와정도관리

BACKGROUND: Point-of-care testing (POCT) glucometers are widely being used for management of diabetes. We examined the analytical performance of the recently developed glucometer CareSens(R) N Glucometer (i-SENS Inc., Korea). METHODS: CareSens N was evaluated for linearity, precision, and the effect of hematocrit. Method comparison using the laboratory reference method, hexokinase method by Hitachi 7600 (Hitachi Co., Japan) was also performed. Other glucometers, Accu-Chek(R) inform (Roche Diagnostics LTD., Germany) and Onetouch(R) ultra(TM) (Lifescan Inc., USA) were evaluated for the same categories according to CLSI guidelines. RESULTS: CareSens N Glucometer showed a good linearity and precision. The linearity was r=0.9965. The coefficients of variations (CVs) of within-run precision were 0.73-1.98% and CVs of total precision were 1.65-2.71%. A high correlation (glucose by CareSens N = 0.9767 x glucose by Hitachi 7600 + 4.1734, r=0.9614) was also shown between the CareSens N glucometer and Hitachi 7600 in the central laboratory. Other glucometers showed a good linearity. The within-run and total-run CVs of other glucometers were within 10%. Although differences with the reference method were within allowable ranges, all glucometers showed variable bias compared with the reference method. Overestimation or underestimation of glucose values were observed by change of hematocrit in range of 31.1 to 51.2%. CONCLUSIONS: CareSens N showed good linearity, precision, and correlation with reference method. CareSens N provided reliable result of blood glucose and seems appropriate for clinical use in the management of diabetic patients.

Evaluation of the Blood Gas Analyzer GEM Premier 4000 / 임상검사와정도관리

BACKGROUND: We evaluated the performance of the GEM Premier 4000 (Instrumentation Laboratory, USA), a new blood gas/electrolytes/co-oximetry analyzer, according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. METHODS: Within-run precision, total-run precision, linearity and sample-related carryover were analyzed using quality control materials at three different concentration levels for each analytes. Correlation was compared with the routinely used NOVA CCX2 (Nova Biomedical, USA) with patients' whole blood samples. RESULTS: The within-run and the total-run precisions of the GEM Premier 4000 showed very low CV of 0.04~4.40% and 0.06~4.11%, respectively, in all parameters except the lactate, which had CV of 5.58% in Level 1 QC material. The system showed a good linearity (r2=0.997~1.000, systemic error=0.00~0.20%) for all items. Sample-related carryover was -4.35%~0.15%. In comparison with the NOVA CCX2 instrument, correlation was high in all parameters with the r value ranging from 0.983-0.999 except for carboxyhemoglobin (r=0.804) and methemoglobin (r=0.010) whose concentrations were in the lower level. CONCLUSIONS: GEM Premier 4000 showed good analytical performance required for blood gas analyzer in its precision, linearity, sample-related carryover, and close correlation with NOVA CCX2. It fulfills most of the requirements for both point-of-care and laboratory use.

Evaluation of GLUCOCARD X-METER Glucose Monitoring System / 대한진단검사의학회지

BACKGROUND: Point-of-care testing (POCT) glucometers are increasingly being used for making important therapeutic decisions and managing diabetes. We examined the analytical performance of GLUCOCARD X-METER (ARKRAY Global Business Inc., Japan) against three other glucometers and a reference laboratory method. METHODS: We evaluated the analytical performance of GLUCOCARD X-METER in comparison with three other glucometers. Studies on precision, linearity, the analysis time, and effects of hematocrit and temperature were carried out and the results were compared with those of the laboratory reference method (hexokinase method by Hitachi 760, Hitachi Co., Japan). RESULTS: GLUCOCARD X-METER showed a good linearity and within-run and total-run precision. Comparison between each glucometer and the Hitachi 7600 showed a good correlation. Although differences with the reference method were within an allowable range, all glucometers showed variable bias. Application of an insufficient amount of blood could produce some changes in test results. Changes in hematocrit were found to cause overestimation or underestimation of glucose values. For some test strips, the results were affected by prolonged exposure to room temperature or 4degrees C refrigerator. CONCLUSIONS: GLUCOCARD X-METER showed a good analytical performance in linearity, precision, and comparison. The effect of hematocrit, sample volume, and storage condition for test strips were noted and glucometers had variable deviations to both directions from laboratory reference values (<20%). The GLUCOCARD X-METER provided rapid and reliable measurements of blood glucose. It could be appropriate for monitoring blood glucose values in diabetic patients.

Evaluation of SD BIOLINE Tetanus Kit / 대한진단검사의학회지

BACKGROUND: Point-of-care-testing (POCT) kits for tetanus toxoid antibody are used in emergency departments to evaluate the immunization status of patients with tetanus. The objective of this study was to evaluate the analytical performance and the utility of SD BIOLINE tetanus kit (Standard Diagnostic Inc., Yongin, Korea), as a POCT. METHODS: A total of 326 peripheral blood specimens (whole blood, 319; serum, 326) from healthy subjects and patients were used. SD BIOLINE tetanus kit was evaluated for precision, accuracy, effect of specimens, operator variance, and the total processing time. The results from SD BIOLINE tetanus kit were compared with those from 2 quantitative ELISA kits. RESULTS: Compared with ELISA kits, SD BIOLINE tetanus kit revealed a sensitivity of 88-97%, specificity of 87-92%, positive predictive value of 81-89%, negative predictive value of 90-98%, and kappa agreement of 0.78-0.82. SD BIOLINE tetanus kit also showed an excellent precision and a high accuracy. It showed a high concordance rate between whole blood and serum specimens. The total processing time of SD BIOLINE tetanus kit was 30-40 min. CONCLUSIONS: SD BIOLINE tetanus kit showed an excellent analytical performance. With its rapid turnaround time and the ease of handling and interpretation, SD BIOLINE tetanus kit seems appropriate for the evaluation of tetanus immunization status as a POCT device. However, education for operators and standardized guidelines for result interpretation should be emphasized.

Clinical Evaluation of i-STAT in the Neonatal Intensive Care Unit / 대한진단검사의학회지

BACKGROUND: i-STAT (i-STAT Corporation, Princeton NJ, USA), a hand-held point-of-care testing (POCT) analyzer with rapidity and minimal sample requirement, has the potential to bring about a significant impact on the management of neonates. However, there should be an overall deliberation of the routine use of i-STAT in the neonatal intensive care unit (NICU) as to whether it is technically reliable and cost-effective. The aim of this study was to assess the clinical aspects of the implementation of i-STAT in the NICU. METHODS: We surveyed physicians and nurses to measure the present status of POCT. We ana-lyzed 84 tests performed in the central laboratory, 88 tests by blood gas analyzer in NICU, and 95 tests by i-STAT for NICU patients. We investigated the indications, turnaround time (TAT), cycle time, and impact on patient care in each case during both pre- and post-i-STAT periods. Costs and user acceptability were also examined. RESULTS: Survey responders wanted rapid results but did not accept the responsibility for the quality of POCT. Turnaround time of i-STAT was shorter than that of the central laboratory, but did not make an impact on cycle time. The cost of i-STAT is 2.2 times higher than central laboratory cost, but the users were satisfied with i-STAT mainly because of its small sample volume and speed. Central laboratory testing volume decreased by 14.3% after the introduction of i-STAT. CONCLUSIONS: i-STAT may be acceptable in the NICU setting. However, the behavioral patterns of physicians need to be changed and a selective use of i-STAT is warranted to maximize its cost-effectiveness. Future studies on the clinical outcome are required to substantiate the potential role of i-STAT.

Evaluation of Point-of-care kit for Diagnosis of Malaria in Korea / 대한임상병리학회지

BACKGROUND: Recently malaria infection became one of the most important parasitic diseases in Korea. After the re-emergence of malaria in a young soldier in 1993 near the De-Militarized Zone (DMZ), three to four thousand people have been infected per year in the last few years and the cases of infection have been increasing threefold each year. Microscopic examination of a thick blood smear is a conventional and confirmatory method for diagnosis. However, it requires labor-intensive procedures and its interpretation is quite subjective. Faster and more reliable methods are needed for the diagnosis of malaria. METHODS: We evaluated 155 patients who were diagnosed as malaria. We performed point-of-care rapid diagnostic methods recently introduced: two antibody detection tests manufactured by Korean companies and one antigen (Plasmodium lactate dehydrogenase, pLDH) detection test. The results were compared with those of microscopic examinations of thick blood smears. RESULTS: Sensitivities of two antibody detection assays and one antigen detection assay in acute attacks of malaria were 64.7%, 72.5%, and 96.1%; and, specificities were 88.5%, 89.4%, and 95.1%, respectively. Overall accuracy for all samples were 80.6%, 83.9%, and 95.5%, respectively. CONCLUSIONS: Antibody detection tests for malaria have limitations in sensitivity and accuracy to replace microscopic examination of blood film. Antigen tests detecting pLDH could replace conventional microscopic examinations of blood film, especially in emergency situations in cases that require prompt medication.