Application of NASBA and RPA in detection of pathogenic bacteria / 中华流行病学杂志

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.

Detection of nucleic acid sequence-based amplification products by gold nanoprobe-based solution hybridization for the diagnosis of invasive aspergillosis / 临床检验杂志

Objective To establish a method of gold nanoprobe-based solution hybridization (GNBSH) to detect nucleic acid sequence-based amplification (NASBA) products for the rapid diagnosis of invasive aspergillosis (IA).Methods The Aspergillus specific 18S rRNA was amplified by NASBA and then the amplified products were hybridized with the gold nanoprobes which were modified with thiol compounds at the 5'end.Serum samples from 106 patients,including 14 with a definite IA,32 with suspected IA and 60 without IA,were detected by the established method,and the obtained results were compared with that of galactomannan (GM) test to evaluate its accuracy.Results The gold nanoprobes only hybridized with Aspergillus NASBA products but not other non-Aspergillus strains.The sensitivity,specificity and the area under the ROC curve (AUCROC) of the established GNBSH method for detecting 106 clinical samples were 82.61％ (38/46),81.67％ (49/60) and 0.890,respectively.The sensitivity,specificity and AUCROC of GM test were 56.52％ (26/46),83.33％ (50/60) and 0.723,respectively.Conclusion The established GNBSH method to detect Aspergillus NASBA products has high sensitivity and specificity and simple operation,which may be used to detect the infection of Aspergillus by clinical laboratories.

Technologies for trans-institutional sharing of scientific data / 中华医学图书情报杂志

Technologies for the trans-institutional sharing of scientific data were studied with the sharing of nucleic acid sequence data as the study object , such as leaving a flexible space for implementation of the unified standards in working out the criteria for data sharing , implementing regular point to point data sharing and updating in agree-ment of institutional league , and providing multiple data services according to the unified working process by giving considerations to the local demands .The significance of trans-institutional sharing of scientific data under the insti-tutional league model was summarized for the reference in constructing the scientific datasharing platform.

Diagnostic value of immediate early antigen-mRNA in the diagnosis of cytomegalovirus infection after kidney transplantation / 中国综合临床

Objective To investigate the diagnostic value of the immediate early antigen(IE) mRNA by nucleic acid sequence-based amplification(NASBA) in peripheral blood cytomegalovirus (CMV) infection,and to establish and promote the diagnosis method for CMV.Methods Five to seven ml blood was taken from 32 patients at 3 week and 7 week after renal transplantation to detect serum cytomegalovirus antigen and antibody expression by NASBA,Real time-PCR and enzyme-linked immunosorbent assay (ELISA) sensitivity and specificity were compared.Results The results of CMV detection in 32 renal transplanted patients respectively showed that the positive rate of peripheral blood IE-mRNA by NASBA was 45.8％ (15/32) ;The positive rate of HCMV-DNA in blood by Real time-PCR was 45.8％ (15/32).Using ELISA,the positive rate of HCMV-(IgG +IgM) was 37.5％ (14/32).IE-mRNA and HCMV-DNA had higher sensitivity and specificity and lower false positive rate than HCMV-(IgG +IgM).The positive rates of IE-mRNA by NASBA,Real time-PCR and ELISA were 92.8％,71.5％ and 42.8％ respectively in the 14 cases.Conclusion The nucleic acid amplification method (NASBA based sequence) and Real time-PCR are sensitive,rapid diagnosis methods of HCMV infection,with higher sensitivity and specificity and lower false positive rate than traditional ELISA.And NASBA detection of IE-mRNA has good value for auxiliary clinical diagnosis.

Real-Time Nucleic Acid Sequence-Based Amplification to Predict the Clinical Outcome of Invasive Aspergillosis

Monitoring the response to therapy for invasive aspergillosis (IA) is essential for the management of patients with hematologic diseases. We evaluated the correlation between the outcome of real-time nucleic acid sequence-based amplification (RTi-NASBA) for Aspergillus 18S rRNA and the clinical outcome of IA. A total of 157 serum samples from 29 patients with IA were tested for RTi-NASBA. The treatment response and mortality were compared with the NASBA outcome (whether the NASBA value was converted to negative or not) at 12 weeks after the start of antifungal therapy. At 12 weeks, there was a moderate correlation between the treatment failure and persistently positive NASBA (kappa = 0.482; P = 0.019). Deaths attributable to IA were more prevalent in patients without negative conversion of NASBA than in those with negative conversion (50% vs 5%; P = 0.013). Significant factors of treatment failure at 12 weeks were the status of hematologic disease (nonremission; P = 0.041) and the NASBA outcome (failure of negative conversion; P = 0.024). Survival was significantly better in patients with negative conversion of NASBA than those with persistently positive values (P = 0.036). This study suggests that the serial monitoring of RTi-NASBA could be useful for prediction of the clinical outcome in hematologic patients with IA.

Detection of Enterovirus using Real-Time Nucleic Acid Sequence-based Amplification / 대한임상미생물학회지

BACKGROUND: Enteroviruses are the most frequent etiologic agents of aseptic meningitis and are estimated to be the cause of 70% to 90% of viral meningitis cases. Enterovirus diagnosis can be difficult because clinical features vary according to patient immunity and age. The purpose of this study was to evaluate the performance of the real-time nucleic acid sequence-based amplification (NASBA) assay compared to that of the real-time nested RT-PCR assay for enterovirus detection. METHODS: This study was performed on 96 patients suspected of aseptic meningitis based on clinical features. RNA was extracted using NucliSENS EasyMAG and real-time NASBA assay was performed using NucliSENS EasyQ Enterovirus and NucliSENS EasyQ Basic 2. We also executed in-house real-time nested RT-PCR assay for RNA extracted via QIAamp Viral RNA Mini. RESULTS: The positive rate of real-time NASBA assay was 45.8% for enterovirus detection. The positive rate of first real-time reverse transcription PCR was 22.9% and the second real-time PCR was 57.3%. The concordant rate of the real-time NASBA assay and first real-time reverse transcription PCR was 75.0%. The concordant rate of the real-time NASBA assay and second real-time PCR was 86.5%. CONCLUSION: The detection of enteroviruses using the real-time NASBA assay is less prone to cross-contamination and is simple, without the need for reverse transcription. We conclude that the NASBA assay is an effective method for the rapid diagnosis of aseptic meningitis.

Nucleic Acid Sequence-Based Amplification and Its Applications in Viral Diagnosis / 中国生物工程杂志

Nucleic acid sequence-based amplification(NASBA) is a sensitive,isothermal,transcription-based amplification system specifically designed for the detection of RNA targets,which could amplify templete RNA in 2h under isothermal condition at about 42?C and without any special equipment.NASBA is now widely applicated in diagnosis of many pathogenic microorganism.It is mainly about principles and applications of NASBA in viral diagnosis.

Serotyping and Phylogenetic analysis of Enteroviruses Isolated from Patients with Aspetic Meningitis / 대한임상미생물학회지

BACKGROUND: The determination of serotype of enteroviruses is useful for the discrimination between sporadic and epidemic infections. The conventional serotyping method is time-consuming and labor-intensive. Recently, molecular method was introduced for the serotyping of enteroviruses. The aim of this study was to establish a method to isolate and analyze enteroviruses from various specimens utilizing molecular biological techniques and to determine which strains were phylogenetically related to clinical samples. METHODS: Clinical samples in this study included 164 cerebrospinal fluid (CSF), 136 stool, 15 sera, 6 throat swab, 5 urine, and 4 sputa, which were obtained from hospitalized patients, primarily infants or children presenting symptoms of aseptic meningitis in 1998. RD cells were used for enterovirus isolation. RT-PCR was performed with RD cell lysate showing CPE. The primers 011 and 012 were used for the VP1 region, and the primers EN1 and EN2 for 5'-UTR. The nucleotide sequences of VP1 region were determined and analyzed with BLAST program. RESULTS: Among 333 samples, only 23 samples produced CPE: 17 samples at first and six samples at the second blind passage. Fifteen isolates were related to coxsackievirus B2 two to echovirus 4, three to echovirus 6, and three to echovirus 18. All 23 viral isolates displayed a nucleotide sequence identity of 80-95%, compared with the reference serotypes. However, the identity was increased up to 93-100% when the VP1 region was translated into amino acids CONCLUSIONS: Since CB2 type was 55% among enteroviral isolates, the CB2 was determined as the major causative serotype of enteroviral meningitis in 1998. CB2 type was emerged between June and July, EC4 and EC6 was limited to July, and EC18 was in August.

Serotyping and Phylogenetic analysis of Enteroviruses Isolated from Patients with Aspetic Meningitis / 대한임상미생물학회지

BACKGROUND: The determination of serotype of enteroviruses is useful for the discrimination between sporadic and epidemic infections. The conventional serotyping method is time-consuming and labor-intensive. Recently, molecular method was introduced for the serotyping of enteroviruses. The aim of this study was to establish a method to isolate and analyze enteroviruses from various specimens utilizing molecular biological techniques and to determine which strains were phylogenetically related to clinical samples. METHODS: Clinical samples in this study included 164 cerebrospinal fluid (CSF), 136 stool, 15 sera, 6 throat swab, 5 urine, and 4 sputa, which were obtained from hospitalized patients, primarily infants or children presenting symptoms of aseptic meningitis in 1998. RD cells were used for enterovirus isolation. RT-PCR was performed with RD cell lysate showing CPE. The primers 011 and 012 were used for the VP1 region, and the primers EN1 and EN2 for 5'-UTR. The nucleotide sequences of VP1 region were determined and analyzed with BLAST program. RESULTS: Among 333 samples, only 23 samples produced CPE: 17 samples at first and six samples at the second blind passage. Fifteen isolates were related to coxsackievirus B2 two to echovirus 4, three to echovirus 6, and three to echovirus 18. All 23 viral isolates displayed a nucleotide sequence identity of 80-95%, compared with the reference serotypes. However, the identity was increased up to 93-100% when the VP1 region was translated into amino acids CONCLUSIONS: Since CB2 type was 55% among enteroviral isolates, the CB2 was determined as the major causative serotype of enteroviral meningitis in 1998. CB2 type was emerged between June and July, EC4 and EC6 was limited to July, and EC18 was in August.