Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.
DNA, Single-Stranded ; Nucleic Acid Amplification Techniques

BACKGROUND: Following discontinuation of the recombinant immunoblot assay (RIBA), the only available supplementary test for the detection of hepatitis C virus (HCV) is the nucleic acid amplification test (NAAT). However, the NAAT does not adequately detect past HCV. Consequently, it is hard to distinguish between past HCV infection and biological false positivity with an anti-HCV result alone. We assessed the diagnostic performance of two immunoassays: the ARCHITECT anti-HCV chemiluminescent microparticle immunoassay (CMIA; Abbott Diagnostics, Wiesbaden, Germany) and the Access HCV Ab PLUS chemiluminescent immunoassay (CIA; Bio-Rad, Marnes-la-Coquette, France). We also explored an optimized algorithm to determine the anti-HCV results. METHODS: We tested 126,919 patients and 44,556 individuals who underwent a medical checkup. RIBA and NAAT were conducted for samples that tested anti-HCV-positive using CMIA and CIA. We assessed the optimal signal-to-cutoff (S/CO) ratio in HCV-positive samples. RESULTS: In total, 1,035 blood samples tested anti-HCV-positive. Of these, RIBA was positive in 512, indeterminate in 160, and negative in 363 samples. One hundred sixty-five samples were NAAT-positive. Diagnostic sensitivity and positive predictive value (PPV) were 96.7% and 52.1%, respectively, for CMIA, and 94.7% and 72.3%, respectively, for CIA. The optimal S/CO ratio was 5.2 for CMIA and 2.6 for CIA at 95% PPV. In total, 286 samples tested positive in CMIA and 444 in CIA, while 443 samples tested positive in both assays. CONCLUSIONS: It is hard to determine anti-HCV positivity based on the S/CO ratio alone. However, this study elucidated the role of the S/CO ratio by using the NAAT and RIBA.
Hepacivirus ; Humans ; Immunoassay* ; Nucleic Acid Amplification Techniques*

In this study, the molecular marker technology of SRAP and ISSR were applied in rapid identification of seeds from eight species of Brassica oleracea L. Firstly, using the genomic DNA of cabbage as template, SRAP and ISSR reaction systems were optimized through testing every factor, respectively, that affects PCR amplification. Then, using the optimized reaction systems, 30 SRAP primer pairs and 15 ISSR primers were applied to amplify genomic DNA of cabbage, savoy, purple cabbage, borecole, cauliflower, broccoli, Brussels sprouts, and kohlrabi The results showed that high polymorphisms were exhibited among the eight species of Brassica oleracea L. by SRAP primer pairs of M3-E5 and M4-E5, as well as ISSR primers of 844 and 888, especially primer 844 which can identify all eight materials efficiently.
Brassica ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Polymorphism, Genetic ; Repetitive Sequences, Nucleic Acid ; Seeds ; genetics

Prevalence of HIV infection in the Philippines remains low. This may be partly due to reliance on passive reporting for surveillance. The algorithm for laboratory testing for HIV infection has become more stringent in the sense that the screening assays are repeated in the confirmatory centers, before Western Blot is performed. This has been due to the high rate of false positives before 2005. Nucleic Acid Amplification testing (NAAT) has been performed routinely for blood banking purposes in other countries. In a few pilot studies, it has proven useful in identifying those cases in the early stage of the infection, which are missed on testing by antibody-based assays. The assay may prove useful in knowing whether false negatives happen with the current testing algorithm in the Philippines. Coupled with the detuned assay, identification of new cases may be critical for prevention of transmission, surveillance of cases, and early medical management if needed.

In May 2015, we conducted a voluntary online survey on laboratory diagnostic assays for Clostridium difficile infection (CDI) across clinical microbiology laboratories in Korea. Responses were obtained from 66 laboratories, including 61 hospitals and five commercial laboratories. Among them, nine laboratories reported having not conducted CDI assays. The toxin AB enzyme immunoassay (toxin AB EIA), nucleic acid amplification test (NAAT), and C. difficile culture, alone or in combination with other assays, were used in 51 (89.5%), 37 (64.9%), and 37 (64.9%) of the remaining 57 laboratories, respectively, and 23 (40.4%) of the laboratories performed all three assays. Only one laboratory used the glutamate dehydrogenase assay. Nine laboratories used the toxin AB EIA as a stand-alone assay. The median (range) of examined specimens in one month for the toxin AB EIA, NAAT, and C. difficile culture was 160 (50–2,060), 70 (7–720), and 130 (9–750), respectively. These findings serve as valuable basic data regarding the current status of laboratory diagnosis of CDI in Korea, offering guidance for improved implementation.
Clinical Laboratory Techniques ; Clostridium difficile ; Clostridium ; Glutamate Dehydrogenase ; Immunoenzyme Techniques ; Korea ; Nucleic Acid Amplification Techniques

Genetic Techniques ; Genomics ; Humans ; Nucleic Acid Amplification Techniques ; Nucleic Acid Hybridization ; Sequence Analysis, DNA ; Viruses ; genetics ; isolation & purification

Bacillus licheniformis alpha-amylase (BLA) is one of the most important enzymes involved in starch hydrolysis and many biotechnological processes. To improve the BLA productivity, an integrative plasmid pBL-amyL carrying amyL gene encoding a thermophilic alpha-amylase of B. licheniformis was constructed and transformed into B. licheniformis B0204, an industrial alpha-amylase producer. The transformants harboring different copies of amyL were developed on kanamycin by using homolog-mediated chromosomal amplification of alpha-amylase gene. The recombinants with different multiple copies of amyL integrated in the chromosome were identified by real-time PCR and evaluated by shake-flask fermentation. Recombinants harboring 2-5 multiple copies of amyL produced more alpha-amylase comparison to the parental strain B0204.
Bacillus ; enzymology ; genetics ; Gene Amplification ; Industrial Microbiology ; Nucleic Acid Amplification Techniques ; Transformation, Genetic ; alpha-Amylases ; biosynthesis ; genetics

OBJECTIVETo establish and evaluate the methods of internal quality control in blood donor screening by nucleic acid test (NAT).

METHODSAfter HBV-DNA standard quality control (QC) sample (60 IU/ml) was diluted by pooling 6 samples, the concentration was 10.0 IU/ml, which was approach twice of the low limit. When the pooling result turned out reactive, the pooling samples need to be split into single sample to process. Meanwhile, the standard QC samples were tested as well. The same batch QC samples were tested 20 times respectively, calculate the mean (x̄), standard deviation (SD) and CV. Make Levey-Jennings QC curves by setting x̄±2SD as warning, x̄±3SD as rejected. The Levey-Jennings quality controls chart was mapped by using Microsoft Excel.

RESULTSAfter 20 times test of mixed/split samples, the x̄±2SD were 33.03±1.47 and 30.08±0.98, the x̄±3SD were 33.03±2.20 and 30.08±1.47, the CV were 2.22% and 1.63%, respectively. The P value of t test was 0.08 and 0.17 respectively, there was no statistically significant difference between the 2 group.

CONCLUSIONWhen establish an internal QC system in the screening laboratory by nucleic acid testing, the concentration of the QC samples should be equal to normal specimens. This type of QC system may validate the extraction and amplification of the nucleic acid, and improve the stability of the test results.

BACKGROUND: Real-time PCR has been widely used not only for quantification?of disease-related genes but also for detection of bacteria or viruses. In this study, we evaluated the performance of Artus HBV LC PCR kit (Qiagen, Hilden, Germany) using recently developed SLAN real-time PCR detection system (Shanghai Hongshi Medical Technology Co., Shanghai, China) to assess clinical relevance of the new instrument. METHODS: Precision, linearity and detection limit of Artus HBV LC PCR kit were evaluated using SLAN real-time PCR detection system. We also compared the SLAN real-time PCR detection system with LightCycler 1.5 (Roche Molecular System, Branchburg, NJ, USA) and ABI PRISM 7500 (Applied Biosystems, Foster City, CA, USA). WHO International Standard for HBV DNA Nucleic Acid Amplification Techniques (NIBSC code:97/750) and 40 HBV DNA positive sera were tested for this evaluation. RESULTS: Within-run and between-day coefficients of variation were 5.63%, 4.01% at 6.2x10(3) IU/mL and 1.12%, 0.80% at 2.1x10(1) IU/mL, respectively. Linearity was verified from 1.0x10(1) to 1.0x10(5) IU/mL (r(2)=1.000; slope=1.1412). Detection limit for HBV DNA was verified to be 7.54 IU/mL. It showed a good correlation with LightCycler 1.5 (r=0.9723) and ABI PRISM 7500 (r=0.9768). CONCLUSIONS: Artus HBV LC PCR kit using SLAN real-time PCR detection system showed a good precision, linearity and assay sensitivity. It correlated well with LightCycler 1.5 and ABI PRISM 7500. We conclude that it can be used in clinical laboratories for nucleic acid quantification.
Bacteria ; DNA ; Limit of Detection ; Nucleic Acid Amplification Techniques ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction

OBJECTIVETo establish a simple and rapid molecular detection for Legionella pneumophila.

METHODSThe loop-mediated isothermal amplification (LAMP) was applied for detection of Legionella pneumophila. A set of primers were designed to identify six special areas in mip gene of Legionella pneumophila. Genomic DNAs from 13 bacterial strains,including 8 Legionella pneumophila strains and 5 other bacterial strains were amplified by LAMP and general PCR method to evaluate the specificity and sensibility of LAMP.

RESULTAll positive tubes produced visible white precipitation, and no precipitation was observed in others. By adding smart green fluorescent dye, all Legionella pneumophila positive tubes presented a strong green fluorescence, while others showed weak fluorescence. The detection rate of LAMP was higher than that of general PCR. The detection limits were 576fg with genomic DNA of Legionella pneumophila,and 8 cfu/mL with positive water samples.

CONCLUSIONLAMP detection of Legionella pneumophila is an effective and low-cost method with high specificity and sensitivity requiring no special equipment.