Simultaneous Amplification And Testing Research Articles

Isothermal Amplification-Based Detection of Single-Base RNA N6-Methyladenosine.

N6-methyladenosine (m6A) has recently gained much attention due to its diverse biological functions. Currently, the commonly used detection methods for locus-specific m6A marks are complicated to operate, it is difficult to quantify the methylation level, and they have high false-positive levels. Here, we report a new method for locus-specific m6A detection based on the methylate-sensitive endonuclease activity of MazF and the simultaneous amplification and testing (SAT) method, termed "m6A-MazF-SAT". Mechanically, MazF fails to cleave the A (m6A) CA motif; therefore, the undigested template can be SAT-amplified using specific probes targeting the upstream and downstream of sites of interest. Fluorescent signals of SAT amplification can be detected by real-time PCR, and therefore, they achieve the detection of m6A existence. After the condition optimization, m6A-MazF-SAT can significantly, accurately, and rapidly detect the m6A-modified sites in mRNA, rRNA, and lncRNA at the fmol level, as well as 10% m6A at the fmol level. In addition, m6A-MazF-SAT can quantify the abundance of target m6A in biological samples and can be used for the inhibitor selection of m6A-related enzymes. Together, we offer a new approach to detect locus-specific m6A both qualitatively and quantitatively; it is easy to operate, results can be obtained rapidly, and it has low false-positive levels and high repeatability.

Evaluation of reverse transcription-polymerase chain reaction and simultaneous amplification and testing for quantitative detection of serum hepatitis B virus RNA

BackgroundChronic hepatitis B virus (HBV) infection is one of the common infectious diseases in the world. HBV covalently closed circular DNA (cccDNA) is the initial template of HBV replication, which can exist in human hepatocytes for a long time and is difficult to be completely removed. It has been shown that HBV RNA can directly respond to the levels and transcriptional activity of cccDNA in hepatocytes and can be used as a surrogate marker of cccDNA transcriptional activity. At present, the detection techniques used for quantitative HBV RNA mainly include reverse transcription quantitative polymerase chain reaction (RT-qPCR) and simultaneous amplification and testing (SAT). MethodsIn this study, we verified the performance of the SAT method for detecting HBV RNA and the clinical effectiveness of SAT and RT-qPCR, and compared the correlation and consistency of the two detection methods for HBV RNA detection. ResultsThe results showed that the limit of detection for HBV RNA by SAT method was 50 copies/mL, with a linear range of 1 × 102-1 × 108 copies/mL. There was no difference in HBV RNA levels detected by the two methods. The correlation and consistency of the results were good, with the coefficient of determination of 0.7787 in HBeAg positive group and 0.8235 in HBeAg negative group. ConclusionsTherefore, this study confirmed that the SAT method and RT-qPCR for detecting HBV RNA have good agreement, which are both reliable methods to detect HBV RNA and can replace each other.

Evaluation of the performances of InnowaveDx MTB-RIF assay in the diagnosis of pulmonary tuberculosis using bronchoalveolar lavage fluid

ObjectiveTo evaluate the tuberculosis diagnostic performance of the InnowaveDx MTB-RIF assay (InnowaveDx test) in bronchoalveolar lavage fluid (BALF). MethodsA total of 213 BALF samples from suspected PTB patients were analyzed. AFB smear, culture, Xpert, Innowavedx test, CapitalBio test and simultaneous amplification and testing (SAT) were performed. ResultsOf the 213 patients included in the study, 163 were diagnosed with PTB, and 50 were TB negative. Using the final clinical diagnosis as the reference, the sensitivity of InnowaveDx assay was 70.6%, which was significantly higher than the values achieved using the other methods (P < 0.05), and the specificity was 88.0%, which was comparable with other methods (P > 0.05). Among the 83 PTB cases with negative culture results, the detection rate of InnowaveDx assay was significantly higher than those of AFB smear, Xpert, CapitalBio test and SAT (P < 0.05). Kappa analysis was used to compare the agreement of InnowaveDx and Xpert in detecting RIF sensitivity, and the result showed the Kappa value was 0.78. ConclusionsThe InnowaveDx test is a sensitive, rapid and cost-effective tool for PTB diagnosis. In addition, the sensitivity of InnowaveDx to RIF in samples with low TB load should be interpreted with caution in light of other clinical data.

Analysis of Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma genitalium and Neisseria gonorrhoeae infections among obstetrics and gynecological outpatients in southwest China: a retrospective study

BackgroundThe aim of this study was to analyze the present situation of Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Neisseria gonorrhoeae (NG) infections among obstetrics and gynecological outpatients in southwest China.MethodsA total of 3225 urogenital swabs were included in this study. All swabs were tested by RNA-based simultaneous amplification and testing (SAT) methods. Routine analysis of leucorrhea smear and drug susceptibility were performed in UU positive patients.ResultsOf these 3225 outpatients, the positive rate was 27.07% for UU, 4.99% for CT, 3.10% for MG, and 0.09% for NG. UU, CT, and MG infections were more common in women of reproductive age (aged 25–34 years), while NG infection was more prominent in women aged 30–34 years and over 40 years. Overall, the infection rate of UU was significantly higher than that of the other three infections, and UU also played a major role even in the mixed infections. 65.07% of the UU positive patients had negative results on routine leucorrhea smear analysis, and the remaining patients may have bacterial vaginitis (15.79%), fungal vaginitis (11.48%), trichomonas vaginitis (0.96%) or other vaginal inflammation (6.70%). We have observed that maternal UU infection can lead to low birth weight, neonatal pneumonia, and premature delivery. The results of the drug susceptibility test of UU showed a higher sensitivity to pristinamycin, doxycycline, tetracycline, clarithromycin, and josamycin (100%, 97.0%, 96.4%, 95.9%, and 95.3%, respectively), and lower sensitivity to ciprofloxacin and ofloxacin (2.4% and 4.7% respectively).ConclusionsThis study could contribute to a better understanding of the current epidemiological features of UU, CT, MG, and NG among obstetrics and gynecological outpatients in southwest China, and thus facilitate to development of the more effective intervention, prevention, and treatment of reproductive tract infection.

Application of real-time simultaneous amplification and testing method to accurately and rapidly detect extra-pulmonary tuberculosis

BackgroundThis study aimed to establish and evaluate a simultaneous amplification and testing method for detection of extra-pulmonary tuberculosis (EPTB).MethodsFrom January 2016 and December 2017 the pus or surgical excision from the lesions of inpatients admitted from Chongqing Public Health Treatment Center were collected. According to the clinical diagnosis, the samples were divided into two groups including EPTB (Group A) and other diseases excluded from tuberculosis diseases (Group B). Simultaneous detection of Mycobacterium tuberculosis (MTB) used Roche culture method, liquid culture method and simultaneous amplification and testing (SAT) method. The sensitivity and specificity of the SAT method were compared with culture methods and clinical diagnosis of EPTB.ResultsFor 433 EPTB specimens and 49 non-TB specimens, the simultaneous amplification and testing tuberculosis (SAT-TB) results correlated with 80.5% (388/482 specimens) of the culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of EPTB were 83.6, 79.4, 59.4, and 93.0%, respectively, compared to culture methods. Compared with the clinical diagnosis of patients, the sensitivity and specificity of the SAT-TB test were 41.6 and 100%, respectively, the cultures test were 29.3 and 98.0%.ConclusionsSAT test is a simple and rapid test with high specificity which may enhance the detection of EPTB. SAT-TB is a higher clinical diagnosis value for EPTB in clinical microbiology laboratories.

Early Diagnosis of Mycoplasma pneumoniae in Children: Simultaneous Amplification and Testing (SAT) Is the Key.

Objective: The effective diagnosis of Mycoplasma pneumoniae (MP) pneumonia (MPP) in children has been hampered by the difficulty of achieving an early diagnosis. The simultaneous amplification and testing (SAT) has the potential for early diagnosis of MP in children.Methods: Of the 1,180 children enrolled in this study, 169 were MPP antibody (Ab) seroconversion positive, 641 showed MPP positivity with a single Ab test, and 370 were MPP negative. Sera and pharyngeal swabs were collected for antibody testing and SAT detection, respectively, on admission. When the samples were Ab negative, the paired -Ab test was requested for MP 7 days later.Results: Using the Ab results as the diagnostic standard, the sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) for SAT were 72.8, 95.1, 97.0, and 61.5%, respectively. SAT had superior diagnostic value in the MPP group who had undergone Ab seroconversion (sensitivity: 82.2%; NPV: 92.1%) and in the short-course group also (sensitivity: 81.0%; NPV: 81.3%). Good agreement was observed between SAT and the paired-Ab results (kappa value = 0.79; P < 0.001), but there was a lack of consistency between SAT and the single-Ab test results on admission (kappa value = 0.54, P < 0.001).Conclusions: SAT is a rapid, sensitive, and specific method for MP diagnosis in pediatric patients. Our results indicate its value as an effective diagnostic tool for detecting MPP at the initial stage of an infection.

Rapid Detection of Mycobacterium Tuberculosis in Lung Tissue and Analysis of Drug Resistance

This study investigated the methods for optimizing the workflow for improving the diagnostic efficiency for detection of Mycobacterium tuberculosis (MTB) in lung tissue specimens. A total of 278 specimens were used in this study. M. tuberculosis in fresh lung tissue samples was detected by BACTEC MGIT 960 culture system, culturing L-form MTB, rifampicin (RFP) and levofloxacin (LVFX) susceptibility test, and ribonucleic acid (RNA) simultaneous amplification and testing (SAT). Specimen samples were embedded in paraffin and serially sectioned. The sections were subjected to Ziehl-Neelsen staining and Intensified Kinyoun (IK) acid-fast staining. The suspected MTB or L-form MTB specimens were further investigated by deoxyribonucleic acid (DNA) sequencing and fluorescence polymerase chain reaction (PCR) melting curve method to detect the mutations in rpoB gene and gyrA gene. Thirteen specimens were suspected as MTB positive, and 37 specimens were suspected to be L-form MTB positive by Ziehl-Neelsen staining and IK acid-fast staining. Among the 50 specimens, the number of MTB positive specimens detected by SAT, DNA sequencing, and fluorescence PCR melting curve method was 43, 44, and 45, respectively. Only 11 MTB positive specimens were detected by BACTEC MGIT 960 culture system or by culturing L-form MTB. Mutations detected in rpoB gene and gyrA gene by fluorescence PCR melting curve method were similar to those detected by DNA sequencing. Some specimens, detected by melting curve method, exhibited varied drug resistance to RFP, suggesting heterogeneous resistance. Among the remaining 228 specimens, there was no MTB or L-form MTB detected by BACTEC MGIT 960 culture system. However, 5 specimens were detected to be MTB positive by the SAT method. The fluorescent PCR melting curve method, which has a high level of automation and high sensitivity and specificity, could effectively detect heterozygous drug resistance of MTB in lung tissue samples, which is important for clinicians to effectively formulate a therapeutic strategy.

Accuracy of Commercial Molecular Diagnostics for the Detection of Pulmonary Tuberculosis in China: A Systematic Review

This systematic review assesses the accuracy of molecular diagnostic methods for the detection of pulmonary tuberculosis in studies performed in China, published in Chinese and English. We searched for studies that assessed the accuracy of molecular diagnostics for pulmonary TB in China in the China National Knowledge Infrastructure, the Wanfang Database, SinoMed, VIP Information, Pubmed, Embase, and the Cochrane Library. For each index test, a summary estimation for sensitivity and specificity was calculated using the bivariate random-effects model. A total of 59 studies were included in our analysis. Loop-mediated isothermal amplifcation (LAMP) assay (six studies; pooled sensitivity 90%, 95% CI 78–95%; specificity 93%, 85–97%), line probe assay (LPA) (one study; 87%, 84–90%; 94%, 92–95%) and polymerase chain reaction (PCR) (FQ-PCR and RT-PCR) (four studies; 90%, 55–99%; 93%, 71–99%) showed good diagnostic performance in the meta-analysis. The highest pooled sensitivity was from Xpert MTB/RIF (20 studies; pooled sensitivity 91%, 95% CI 87–94%). The highest pooled specificity was from cross-priming amplification (CPA) (six studies; pooled specificity 97%, 95–99%). The lowest pooled sensitivity and specificity were from simultaneous amplification and testing (SAT)-TB (three studies; 79%, 66–88%; 72%, 48–88%). In subgroup analysis, molecular diagnostics demonstrated higher sensitivity for pulmonary TB detection in smear-positive specimens. Xpert MTB/RIF, LAMP, LPA, CPA and PCR demonstrated high accuracy overall for pulmonary tuberculosis detection, while SAT-TB had poor performance.

Comparison of rRNA-based and DNA-based nucleic acid amplifications for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum in urogenital swabs

BackgroundNucleic acid amplification tests (NAAT) are well-accepted in diagnosis and surveillance of sexually infectious pathogens worldwide. However, performance differences between a RNA-based NAAT and DNA-based NAAT are rarely reported. This study compares the performances of the RNA-based SAT (simultaneous amplification and testing) assay and the DNA-based quantitative real-time polymerase chain reaction (qPCR) assay.MethodsA total of 123 urogenital swabs were collected from outpatients with suspected genital infections in our hospital. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) in these swabs were simultaneously tested by SAT and qPCR. Any swabs were positive in the qPCR assay were further verified by following cloning and sequencing. All statistical analysis was performed using the SPSS software.ResultsWhen the concentrations of CT, NG, or UU were more than 1 × 103 copies/ml, 100% agreements between SAT and qPCR were observed regardless of the pathogen. No discrepancy was found. However, the sensitivity of SAT is significantly higher than qPCR in samples with concentration less than 1 × 103 copies/ml. When tested by SAT and qPCR, 57.14 and 28.57% were positive for CT, 46.15% and 0 were positive for NG, 80% and 0 were positive for UU, respectively.ConclusionsThe SAT assay has better agreements and higher sensitivities when compared with the qPCR assay, and thus could be a better choice for screening, diagnosis, and surveillance of sexually transmitted diseases, especially for CT and NG.

Detection and clinical application of the isothermal amplification of Mycoplasma pneumonia RNA in bronchoalveolar lavage fluid from children

Objective To assess the ability of simultaneous amplification and testing (SAT) in bacteria detection, bronchoalveolar lavage fluid from children was collected and detected by SAT, combined with in vitro culture experiment, providing theoretical support for the application of SAT in the diagnosis and treatment of mycoplasma pneumonia (MP). Methods A total of 572 bronchoalveolar lavage fluid from children with community acquired pneumonia during October 2015 and December 2017 in Tianjin Children′s Hospital were collected and detected by Mycoplasma pneumonia(MP)nucleic acid quantitative assay and SAT technology. Among them, 161 bronchoalveolar lavage fluid were also detected using in vitro culture and the positive ones for MP were analyzed by nucleic acid quantification and SAT after exposing to high concentration of antibiotic. Results The positive rates of MP by nucleic acid quantitative assay and SAT technology in 572 samples were 74.7% (427/572) and 71.9% (411/572), respectively. These two detection methods have high consistency (χ2=1.142, P=0.285). According to the test results of SAT, the positive rates of male and female were 72.7% (224/308) and 70.8% (187/264), respectively. There was no significant difference of positive rate between different sex (χ2=0.252, P=0.616). The positive rate of MP in 4-14 years old children (78.1%, 317/416) was higher than that in infants (≤3 years) (56.6%, 94/166) (χ2=26.811, P=0.000). After adding azithromycin (5 MIC) to MP positive medium for 5 days, the result of nucleic acid quantification was positive but SAT was negative. Conclusions SAT technology is a rapid, sensitive and specific method for detection of MP. In addition, SAT technology could identify the dead and live bacteria and could evaluate the effect of clinical treatment effectively.(Chin J Lab Med, 2018, 41: 770-774) Key words: Mycoplasma pneumoniae; Bronchoalveolar lavage fluid; Nucleic acid amplification techniques; Microbial sensitivity tests

Portable Infrared Isothermal PCR Platform for Multiple Sexually Transmitted Diseases Strand Detection.

Rapid and portable PCR detection is essential for screening sexually transmitted infections regularly. We developed an infrared mediated RNA isothermal RT-PCR (IR-MERIT PCR) platform and its compatible multichamber microfluidic chip for simultaneous amplification and testing (SAT) detection. This microfluidic chip integrates RNA extraction, micropump, and multitarget detection function onto the same chip. By utilizing IR-light-emitting diode (LED) as heat source, this platform can fulfill isothermal amplification within 70 min.

Application of SAT technique in detection of Chlamydia trachomatis and Ureaplasma urealyticum

Objective To study the Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) infection in Guangzhou area, and analyze the consistency of simultaneous amplification and testing (SAT) and conventional methods (CT was detected by latex immunochromatography, UU was detected by liquid culture method). Methods A total of 12 120 samples of urogenital secretions or urine samples were collected from Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University from January 2015 to December 2016. CT-RNA and UU-RNA were detected by the SAT technique, a part of samples were tested by conventional methods at the same time. The positive rates of CT and UU by SAT and the conventional methods between different gender and age groups were analyzed by χ2 test, the consistencies between different detection methods were analyzed by Kappa test. Results The positive rate of CT was 4.05%(356/8 781), UU 33.69%(1 125/3 339)in Guangzhou from 2015 to 2016. The positive rate of UU was significantly higher than that of CT (χ2=1 981, P<0.01). Of 145 specimens for CT test, the coincidence rate between SAT and latex immunochromatographic method was 96.55%(140/145), which showed good consistency (Kappa=0.65). Of 186 specimens for UU test, the coincidence rate of the results between the SAT method and liquid culture was 92.47%(172/186), which showed strong consistency (Kappa=0.81). Conclusions The positive rate of UU was significantly higher than that of CT in Guangzhou. The SAT method and conventional methods to detect CT and UU show high consistency, which can provide the evidence for clinical diagnosis of CT and UU infection.(Chin J Lab Med, 2018, 41: 380-384) Key words: Chlamydia trachomatis; Ureaplasma urealyticum; Bacteriological techniques; Nucleic acid amplification techniques; Immunochromatography

An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification

Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

Evaluation of the RNA simultaneous amplification and testing for detection of rifampin resistance in Mycobacterium tuberculosis

To establish and evaluate a method for detection of rifampin resistance in Mycobacterium tuberculosis (M. tuberculosis) by the RNA simultaneous amplification and testing (SAT). RNA probe and primer of reverse transcription with T7 promoter targets pre-16S rRNA were designed, and the isothermal RNA amplification at 42 °C was performed for real-time detection of the levels of pre-16S rRNA in drug exposed MTB. Twenty clinical isolates were detected by the SAT after treatment with rifampicin 1, 2 and 3 d in order to determine the best drug effect time. Fifty clinical isolates with known drug susceptibility results were used to determine the best cutoff value of rifampicin drug susceptibility by SAT. In total, 128 clinical isolates were detected by SAT to evaluate the accuracy compared with the Bactec MGIT 960. The best drug effect time and cutoff value for the detection of rifampin resistance by SAT were 2 d and 2.95. With the result of Bactec MGIT 960 as the reference, the sensitivity and the specificity of the assay was 100% (51/51) and 97.4% (75/77), respectively. The isothermal RNA amplification assay is a highly sensitive and specific tool for the detection of rifampicin resistance in M. tuberculosis, and therefore, it may be a new method to detect rifampicin resistance in M. tuberculosis.

Development and evaluation of a real-time method of simultaneous amplification and testing of enterovirus 71 incorporating a RNA internal control system

Human enterovirus 71 (EV71) is a primary etiological agent of hand, foot, and mouth disease (HFMD). The associated infections have posed a serious threat to the public health. In the present study, a real-time simultaneous amplification and testing (SAT) technology was developed for detecting EV71 (SAT-EV71). The RNA of EV71 and an internal control (IC) were amplified and analyzed simultaneously by isothermal amplification and real-time detection of fluorescence using routine real-time PCR. Furthermore, this SAT-EV71 method with IC was evaluated by analyzing 256 clinical specimens including 60 enterovirus-positive ones confirmed by the virus-cell culture method. The other 196 putative ones were further analyzed by a PCR-fluorescence probing assay. The results showed that SAT-EV71 can detect the EV71 VP1 with a minimum of 10 copies per reaction at an optimal concentration of IC (5000 copies per reaction) with a high sensitivity and specificity. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. Overall, this easy-to-perform SAT-EV71 with IC can detect EV71 sensitively and specifically. It might be used in the molecular diagnosis of EV71 in putative cases of HFMD.

A new accurate assay for Coxsackievirus A 16 by fluorescence detection of isothermal RNA amplification

Coxsackievirus A 16 (CA16) is one of the most common causes of hand, foot, and mouth disease (HFMD) worldwide. Without a vaccine or antiviral drug early, rapid, and accurate detection is critical for preventing and controlling HFMD. A simultaneous amplification and testing (SAT) assay was developed for detecting CA16 based on isothermal RNA amplification with fluorescence using standard, real-time PCR equipment. Primers and probes were designed to target the VP1 region of CA16. Virus strains and clinical specimens were used to evaluate the diagnostic performance characteristics of the assay. The assay detected as few as 10 copies of CA16 RNA transcripts. Using real-time PCR plus sequencing as the reference standard, the sensitivity and specificity of the SAT-CA16 assay were 100% and 99.2%, respectively. These findings indicate that SAT-CA16 is a rapid and reliable method for detecting CA16.

OL-053 Rapid direct detection of Mycobacterium tuberculosis using Simultaneous Amplification and Testing (SAT)

OL-053 Rapid direct detection of Mycobacterium tuberculosis using Simultaneous Amplification and Testing (SAT)

Clinical analysis of simultaneous amplification and testing technology in the detection of Neisseria gonorrhoeae from genitourinary specimens

Objective To evaluate the performance of simultaneous amplification and testing (SAT) kit for detecting Neisseria gonorrhoeae(NG) from swab and urine specimens. Methods A total of 205 clinical samples of male urethral swab, female endocervical swab and FCU (first-catch urine) were collected from symptomatic and asymptomatic STD clinic clients. The swab samples were tested by both SAT and culture,while FCU samples by SAT only. The samples with inconsistent test results were further re-tested by another PCR. Results NG-SAT yielded the sensitivity and specificity of 100% and 94.5%, respectively, for both swab and urine samples. Six of 7 specimens with divergent results between SAT and culture showed consistent results between SAT and PCR. Conclusion These results indicate that SAT is sensitive, specific, and timesparing in the detection of NG, which may provide a novel approach to the laboratory diagnosis of gonorrhea. Key words: Neisseria gonorrhoeae; Nucleic acid amplification technique; Urine