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

OBJECTIVESTo establish a Chinese national standard for a nucleic acid test (NAT) for HBV DNA.

METHODSThe candidate sample of HBV DNA positive plasma was diluted with HBV-negative human plasma. The sample was lyophilised with a concentration of approximately 300,000 copies/ml. The measurement methods used included Roche Amplicor assay (version 2.0) and real-time PCR. The lyophilised preparation was calibrated by the international standard (NIBSC code: 97/746) from NIBSC.

RESULTSThe quantity of this lyophilised preparation was (1.29+/-0.24) x 10(5)IU/ml in comparison with the international standard for HBV DNA 97/746. The stability test indicated that the sample was stable at room temperature (20 to 25 degrees C) for 2 weeks and at 37 degrees C for at least 1 week. Long-term stability was observed at 2 to 8 degrees C for 6 months and at -20 degrees C for more than 2 years with no significant changes. The vial-to-vial imprecision rate was 3.53%.

CONCLUSIONBased on the results of this study, our lyophilized sample can be used as a standard in China for the nucleic acid test (NAT) for HBV DNA.

OBJECTIVE To assess the application value of multiplex ligation-dependent probe amplification (MLPA) for the detection of gene deletion and prenatal diagnosis of α-thalassemia. METHODS MLPA was applied for 2 cases with α-thalassemia phenotype by whole blood cell counting and hemoglobin component detection but were ruled out by regular molecular diagnosis. Potential gene deletions and point mutations of α-thalassemia gene were detected with regular Gap-polymerase chain reaction (Gap-PCR) and reverse dot blotting (RDB) in 89 cases where one or both partners were carriers of α-thalassemia mutations. Meanwhile, MLPA was used for detecting α-globin gene deletion among the 89 samples. RESULTS For the 2 cases with α-thalassemia phenotype, no α globin gene deletion was detected by MLPA, but were subsequently confirmed as iron-deficiency anemia. The results of MLPA and Gap-PCR detection for the 88 cases were consistent, except for 1 fetal sample (chorionic villi) which could not be diagnosed by Gap-PCR and was confirmed to be - SEA/αα by MLPA. CONCLUSION MLPA can be applied to prenatal diagnosis of α-thalassemia as an effective supplement to Gap-PCR to reduce both misdiagnosis and missed diagnosis and improve the accuracy of prenatal diagnosis.
Adult ; Female ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; alpha-Thalassemia ; diagnosis ; genetics

In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse-blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants.
Nucleic Acid Amplification Techniques ; methods ; Plants, Genetically Modified ; genetics ; Plasmids ; Tobacco ; genetics