CORD-19_Custom_license_subset  

CORD-19:f20529d499463013219363bae1b9af109ae672a9 JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
T1 0-85 Sentence denotes Sensitive and rapid detection of Zika virus by loop-mediated isothermal amplification
T2 87-95 Sentence denotes Abstract
T3 96-212 Sentence denotes Zika virus (ZIKV) is a mosquito-borne flavivirus, which is a pathogen affecting humans in Africa, Asia, and America.
T4 213-314 Sentence denotes It is necessary to detect ZIKV with a rapid and sensitive molecular method to guide timely treatment.
T5 315-589 Sentence denotes In this study, a loopmediated isothermal amplification (LAMP) assay was described, which is an attractive option as a fast, sensitive, and specific method for ZIKV detection using the NS5 protein coding region and the envelope protein (EP) coding region as target sequences.
T6 590-709 Sentence denotes Two different techniques, a calcein/Mn 2+ complex chromogenic method and real-time turbidity monitoring, were employed.
T7 710-776 Sentence denotes The specificity and sensitivity of the LAMP assay were determined.
T8 777-1074 Sentence denotes The assay's detection limit was 0.5 × 10 −9 pmol/µl DNA for NS5 protein coding region and 1.12 × 10 −11 pmol/µl DNA for E coding region, respectively, which is a 100-fold increase in sensitivity compared with real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional PCR.
T9 1075-1210 Sentence denotes All 12 non-ZIKA respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for ZIKV.
T10 1211-1433 Sentence denotes In conclusion, a visual detection LAMP assay was developed, which could be a useful tool for primary quarantine purposes and clinical screening, especially in situations where resources are poor and in point-of-care tests.
T11 1435-1525 Sentence denotes Zika virus (ZIKV) is an emerging mosquito-borne arbovirus of the Flaviviridae family [1] .
T12 1526-1628 Sentence denotes Zika virus has been reported as a pathogen of sporadic human infection for nearly half a century [2] .
T13 1629-1723 Sentence denotes A widespread epidemic of ZIKV infections was found in Central and South America recently [3] .
T14 1724-1875 Sentence denotes On February 1, 2016, the disease has been declared as a "Public Health Emergency of International Concern" by the World Health Organization (WHO) [2] .
T15 1876-1998 Sentence denotes ZIKV is spread via mosquito transmission, sexual transmission, and vertical (mother to child) and blood transfusions [1] .
T16 1999-2225 Sentence denotes The diagnosis of virus infection is typically performed by virus isolation, serological examinations, or specific antibodies from animals, but these methods are time consuming and not appropriate for clinical settings [4, 5] .
T17 2226-2555 Sentence denotes In some virus laboratories, molecular-based diagnostic methods are employed using reverse transcription-PCR (RT-PCR), which takes several hours to obtain the results, which requires special equipment and skillful experiences [5, 6] , and is complicated to perform in resource-limited laboratories in developing countries [7, 8] .
T18 2556-2665 Sentence denotes Therefore, it is important to develop faster, more sensitive, and more accurate methods for timely diagnosis.
T19 2666-2909 Sentence denotes LAMP is an emergency nucleic acid detection technique which was reported in 2000 and it performs high specificity as the amplification of DNA is conducted by two to three pairs of primer recognizing six independent regions on target gene [9] .
T20 2910-3047 Sentence denotes LAMP was developed to amplify target DNA without any temperature change, which typically required denaturation, annealing, and extension.
T21 3048-3235 Sentence denotes As the method could proceed at constant temperature with only a thermostat as the only equipment required, it is an ideal method for use in the field or in time-sensitive situations [9] .
T22 3236-3461 Sentence denotes This technology has been widely used in clinical diagnosis; qualitative and quantitative Edited by Joachim Jakob Bugert. detection of prevalent bacteria, viruses, and parasites; and fetal gender identification, etc. [7, 10] .
T23 3462-3703 Sentence denotes Thus, LAMP could be a highly beneficial and convenient application to detect Zika virus due to its high specificity and stability, and it has been reported that LAMP shined a light on Zika virus [11] [12] [13] [14] [15] [16] [17] [18] [19] .
T24 3704-3846 Sentence denotes The non-structural protein (NS5) and envelope protein (E) coding regions can be used as potential biomarkers for diagnostic purposes [5, 20] .
T25 3847-3938 Sentence denotes Our study is the first to use ZIKV NS5 and E genes as the target sequences in a LAMP assay.
T26 3939-4086 Sentence denotes Here, we describe a visual method based on color change of the calcein/Mn 2+ mixture and LAMP results can be interpreted by naked eyes immediately.
T27 4087-4307 Sentence denotes The purpose of this study was to develop a rapid and simple test of ZIKV detection based on LAMP technology, which requires only basic equipment and interprets results instantaneously through visual inspection. [5, 20] .
T28 4308-4454 Sentence denotes NS5 and E fragments were synthesized (Sangon Biotech Co., Ltd., Shanghai, China) and cloned into pBM21 vector to prepare the recombinant plasmids.
T29 4455-4595 Sentence denotes RNA was extracted from viruses stocks using commercial QIAamp MinElute Virus Spin kit (Qiagen kit) according to the manufacturer's protocol.
T30 4596-4754 Sentence denotes Reverse transcription was conducted according to standard procedure using M-MLV reverse transcriptase (Fermentas) according to the manufacturer's instruction.
T31 4755-4924 Sentence denotes The DNA concentration was determined by measuring the optical density at 260 nm (OD260) and then dissolved in 20 µl of DEPC-treated water and stored at −80 °C until use.
T32 4925-4995 Sentence denotes All infectious materials were handled in biosafety level 3 facilities.
T33 4996-5090 Sentence denotes The NS5 and E sequences of Zika virus strain Natal RGN were chosen as targets for the primers.
T34 5091-5297 Sentence denotes The sequence with accession number KU527068 was retrieved and obtained from NCBI GenBank database, and was further analyzed by Primer Explorer Version 4 (http://prime rexpl orer.jp/elamp 4.0.0/index .html).
T35 5298-5425 Sentence denotes Outer forward primer (F3), outer reverse primer (B3), forward inner primer (FIP), and reverse inner primer (BIP) were designed.
T36 5426-5529 Sentence denotes Two additional loop primers (loop F and loop B) were designed to accelerate the amplification reaction.
T37 5530-5647 Sentence denotes The FIP and BIP primers recognize both sense and antisense strands and were linked by a four-thymidine spacer (TTTT).
T38 5648-5748 Sentence denotes PCR and RT-PCR were performed with the primers named PCR-F, PCR-R, RT-PCR-P, RT-PCR-F, and RT-PCR-R.
T39 5749-5855 Sentence denotes All the primers were synthesized commercially (Sangon Biotech Co., Ltd., Shanghai, China) ( Tables 2, 3 ).
T40 5856-6094 Sentence denotes The LAMP reactions were carried out in a total volume of 25 µl containing 12.5 µl reaction mixture, 1 µl Bst DNA polymerase, 2 µl template by using the Loopamp DNA Amplification kit (Loopamp DNA Amplification Kit; Eiken Chemical Co., Ltd.
T41 6095-6224 Sentence denotes Tochigi, Japan) for real-time turbidimeter and add another 1 µl calcein solution (Eiken Chemical Co., Ltd.) for visual detection.
T42 6225-6306 Sentence denotes An amount of 40 pmol was used for FIP Influenza A H7N9 Our microorganism center 7
T43 6307-6361 Sentence denotes Human coronavirus HCoV-OC43 Our microorganism center 8
T44 6362-6416 Sentence denotes Human coronavirus HCoV-NL63 Our microorganism center 9
T45 6417-6517 Sentence denotes Human coronavirus HCoV-229E Our microorganism center 10 SARS coronavirus Our microorganism center 11
T46 6518-6558 Sentence denotes Dengue virus Our microorganism center 12
T47 6559-6787 Sentence denotes Sudan EBOV (Subtype Sudan, strain Gulu) Our microorganism center 13 ZIKV strain Natal RGN Our microorganism center and BIP; a concentration of 20 pmol was used for LB and LF; and a concentration of 5 pmol was used for F3 and B3.
T48 6788-6869 Sentence denotes Finally, the appropriate amount of template genome is added to the reaction tube.
T49 6870-7023 Sentence denotes The reaction mix was overlaid with the wax to prevent cross-contamination of samples by aerosol and carried out in the reaction tubes (Eiken Chemical Co.
T50 7024-7050 Sentence denotes Ltd.) for 60 min at 65 °C.
T51 7051-7232 Sentence denotes During the amplification process, the wax melted into a liquid without interfering with the reaction, and the wax solidified as the temperature in the tube fell to room temperature.
T52 7233-7346 Sentence denotes Two different detection methods based on sample turbidity and fluorescence were used to detect the LAMP products.
T53 7347-7562 Sentence denotes Spectrophotometric analysis was performed by recording optical density (650 nm) using a Loopamp realtime turbidimeter (LA-320C; Eiken Chemical Co., Ltd.), and real-time changes in turbidity were monitored every 6 s.
T54 7563-7674 Sentence denotes For direct visual detection, 1 µl of calcein fluorescence detection reagent was added to 25 µl of LAMP product.
T55 7675-7784 Sentence denotes As the magnesium ions form a complex with calcein, LAMP amplification produces a green fluorescence emission.
T56 7785-7903 Sentence denotes For negative reactions, the color is still orange, but for positive reactions, the color changes from orange to green.
T57 7904-7993 Sentence denotes It can be observed under natural light or with the naked eye under 365 nm UV light [21] .
T58 7994-8178 Sentence denotes To describe the sensitivity of LAMP detection, a one-step E rRT-PCR assay capable of detecting ZIKV NS5 and E sequences was used with 25 µl reaction mix under the following conditions:
T59 8179-8323 Sentence denotes 2 µl of 5 µM of Probe, 1 µl of 5 µM of each primer, 12.5 µl of 2 × Pre Mix Ex Taq, 7.5 µl of DNA/ RNA free water, and 1 µl of the DNA specimens.
T60 8324-8408 Sentence denotes The negative control consisted of water and twelve genomes of respiratory pathogens.
T61 8409-8598 Sentence denotes The following thermal profile was initially carried out at 94 °C for 5 min; amplification, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s; final extension, 72 °C for 7 min.
T62 8599-8674 Sentence denotes The data were analyzed using the SDS software from Applied Biosystems [5] .
T63 8675-8871 Sentence denotes RT-PCR probe (FAM)ct(c/t)agaccagctgaa(a/g)(BHQ1) Zika(NS5)-qRT-PCR-probe RT-PCR forward aa(a/g)tacacatacca(a/g)aacaaagtggt Zika(NS5)-qRT-PCR-probe RT-PCR backward tcc(a/g)ctccc(c/t)ct(c/t)tggtcttg
T64 8873-9010 Sentence denotes Five and three sets of primers were initially tested to detect ZIKV NS5 gene and E gene under the same reaction conditions, respectively.
T65 9011-9183 Sentence denotes The "Zika 2 (NS5-Nata12)" primer set for NS5 gene and the "Zika-1" primer set for E gene began to amplify the target genes in the shortest time, respectively (Fig. 1a, b) .
T66 9184-9293 Sentence denotes Thus they were chosen as the optimal primer sets for ZIKV NS5 gene and E gene detection with LAMP (Table 2) .
T67 9294-9457 Sentence denotes To optimize the reaction temperatures of the primers in the LAMP reaction, temperatures from 55 to 69 °C at 2 °C intervals were compared for optimal amplification.
T68 9458-9541 Sentence denotes As shown in Fig. 2a, b , the most suitable reaction temperature range was 61-67 °C.
T69 9542-9602 Sentence denotes Finally, we chose 65 °C as the optimal reaction temperature.
T70 9603-10027 Sentence denotes Fig. 1 The most appropriate primers for the loop-mediated isothermal amplification assay. a A total of five sets of primers including Zika(NS5)-2, Zika(NS5)-3, Zika(NS5)-5, Zika1(NS5-Natal1), and Zika2(NS5-Natal2) were designed to detect the recombinant plasmid containing NS5 gene. b A total of three sets of primers includ-ing Zika-1, Zika-10, and Zika-15 were designed to detect the recombinant plasmid containing E gene.
T71 10028-10305 Sentence denotes Turbidity was monitored and recorded every 6 s for five sets of primers used to amplify the target gene with a Loopamp Real-time Turbidimeter at 650 nm Fig. 2 The most appropriate reaction temperatures for the loop-mediated isothermal amplification assay for NS5 (a) and E (b).
T72 10306-10372 Sentence denotes Reaction temperatures ranged from 55 to 69 °C with 2 °C intervals.
T73 10373-10454 Sentence denotes Turbidity was monitored every 6 s with a Loopamp Real-time Turbidimeter at 650 nm
T74 10455-10631 Sentence denotes To test the LAMP specificity for NS5 gene and E gene, we tested 12 non-Zika virus in addition to the recombinant plasmids containing NS5 gene or E gene as the positive control.
T75 10632-10819 Sentence denotes Figure 3 shows that the recombinant plasmids containing NS5 gene or E gene were identified positively by LAMP using turbidity monitoring (Fig. 3a, c) and visual observation (Fig. 3b, d) .
T76 10820-10928 Sentence denotes Figure 3a , b shows the results of NS5 gene detection and Fig. 3c , d shows the results of E gene detection.
T77 10929-11048 Sentence denotes All non-Zika virus tested negative, including the blank control, indicating that the LAMP method was specific for ZIKV.
T78 11049-11200 Sentence denotes To determine the sensitivity of the LAMP assay for ZIKV, a series of dilutions of the recombinant plasmids containing NS5 gene or E gene were prepared.
T79 11201-11592 Sentence denotes The times of positivity detection ranged from 18 min for 0.5 × 10 −4 pmol/µl to 40 min for 0.5 × 10 −9 pmol/µl of the recombinant plasmid containing NS5 gene by real-time monitoring (Fig. 4a ) and the times of positivity detection ranged from 16 min for 1.12 × 10 −6 pmol/µl to 30 min for 1.12 × 10 −11 pmol/ µl of the recombinant plasmid containing E gene by realtime monitoring (Fig. 4d) .
T80 11593-11898 Sentence denotes Thus, the LAMP detection limit for NS5 gene is 0.5 × 10 −9 pmol/µl of the recombinant plasmids containing NS5 gene in a 65 °C reaction lasting for 40 min, and the LAMP detection limit for E gene is 1.12 × 10 −11 pmol/µl of the recombinant plasmids containing E gene in a 65 °C reaction lasting for 30 min.
T81 11899-12049 Sentence denotes For the visual inspection, all positive reactions changed to green while negative ones remained orange under natural or 365 nm UV light (Fig. 4b, e) .
T82 12050-12133 Sentence denotes These data indicate that the sensitivity of the two detection methods was the same.
T83 12134-12190 Sentence denotes For comparison, real-time RT-PCR and PCR were performed.
T84 12191-12343 Sentence denotes The detection limit of real-time RT-PCR for NS5 gene was 0.5 × 10 −7 pmol/µl and was achieved with a higher C t value (C t = 31.68 ± 0.983) ( Table 4 ).
T85 12344-12424 Sentence denotes The detection limit of PCR for NS5 gene was also 0.5 × 10 −7 pmol/µl (Fig. 4c) .
T86 12425-12576 Sentence denotes The detection limit of real-time RT-PCR for E gene was 1.12 × 10 −9 pmol/µl and was achieved with a higher C t value (C t = 32.75 ± 0.085) ( Table 5 ).
T87 12577-12656 Sentence denotes The detection limit of PCR for E gene was also 1.12 × 10 −9 pmol/µl (Fig. 4f) .
T88 12657-12744 Sentence denotes Therefore, we conclude that LAMP assay is more sensitive to ZIKV than real-time RT-PCR.
T89 12745-12958 Sentence denotes ZIKV infection is characterized by mild disease associated with fever, headache, rash, joint pain, and conjunctivitis, and there are special reports related to Guillain-Barré syndrome (GBS) and microcephaly [22] .
T90 12959-13039 Sentence denotes Therefore, rapid and sensitive laboratory diagnostic tests are highly desirable.
T91 13040-13147 Sentence denotes ZIKV's diagnostic tests have traditionally been accomplished through time-consuming methods of cultivation.
T92 13148-13388 Sentence denotes The present study approved the LAMP assay to directly detect Fig. 4 Comparison of the sensitivities of the LAMP reaction and conventional PCR in detecting the ZIKV NS5 gene (a-c). a Turbidity monitoring, b visual detection, c PCR detection.
T93 13389-13502 Sentence denotes The recombinant plasmid containing NS5 gene was serially diluted 10-fold from 0.5 × 10 −2 to 0.5 × 10 −9 pmol/µl.
T94 13503-13739 Sentence denotes 1, negative control (doubledistilled water); 2, 0.5 × 10 −2 pmol/µl; 3, 0.5 × 10 −3 pmol/µl; 4, 0.5 × 10 −4 pmol/µl; 5, 0.5 × 10 −5 pmol/µl; 6, 0.5 × 10 −6 pmol/µl; 7, 0.5 × 10 −7 pmol/µl; 8, 0.5 × 10 −8 pmol/µl; 9, 0.5 × 10 −9 pmol/µl.
T95 13740-13911 Sentence denotes Com-parison of the sensitivities of the LAMP reaction and conventional PCR in detecting the ZIKV E gene (d-f). d Turbidity monitoring, e visual detection, f PCR detection.
T96 13912-14034 Sentence denotes The recombinant plasmid containing E gene was serially diluted 10-fold from 1.12 × 10 −4 pmol/µl to 1.12 × 10 −11 pmol/µl.
T97 14035-14333 Sentence denotes 1, negative control (double-distilled water); 2, 1.12 × 10 −4 pmol/µl; 3, 1.12 × 10 −5 pmol/µl; 4, 1.12 × 10 −6 pmol/µl; 5, 1.12 × 10 −7 pmol/µl; 6, 1.12 × 10 −8 pmol/µl; 7, 1.12 × 10 −9 pmol/µl; 8, 1.12 × 10 −10 pmol/µl; 9, 1.12 × 10 −11 pmol/µl the NS5 and E gene to aid in the diagnosis of ZIKV.
T98 14334-14434 Sentence denotes And a mass of reports proved LAMP had more sensitivity and specificity for detecting pathogen [23] .
T99 14435-14512 Sentence denotes Here, we described a visual LAMP method for detecting NS5 and E gene of ZIKV.
T100 14513-14834 Sentence denotes Detection of specificity was performed here using 13 viruses including 12 of other species and the recombinant plasmid containing NS5 or E gene by real-time turbidimeter and visual methods, respectively, and with the recombinant plasmid containing NS5 or E gene being positive and all the other 12 species being negative.
T101 14835-15011 Sentence denotes These results not only stated that the visual LAMP method has the same specificity with using real-time turbidimeter but also had specificity for detecting ZIKV specific genes.
T102 15012-15225 Sentence denotes Furthermore, we compared the ability of LAMP to detect NS5 and E sensitivity using 10-fold serial dilution of the recombinant plasmid containing NS5 or E gene with that of traditional PCR method and RT-PCR method.
T103 15226-15312 Sentence denotes The detection limit of LAMP assay was 100-fold lower than the value of PCR and RT-PCR.
T104 15313-15380 Sentence denotes More than that, LAMP can finish in 40 min with a simple thermostat.
T105 15381-15520 Sentence denotes Therefore, LAMP posses more rapid and more simple character and these features make LAMP assay more suitable for field and/or time purpose.
T106 15521-15624 Sentence denotes There are various advantages of LAMP assay but it has a relatively high rate of false-positive results.
T107 15625-15920 Sentence denotes For the reason that the amplification efficiency of the LAMP assay is extremely high and 20 µg of specific DNA can be synthesized in a 25-µl reaction mixture within 60 min [24] , it was very necessary to separate the reagent preparation and the performance of the test to avoiding contamination.
T108 15921-16042 Sentence denotes In our study, a low-melting point wax was added to the reaction tube to prevent the spread of the amplification products.
T109 16043-16091 Sentence denotes It seems to work well to avoiding contamination.
T110 16092-16291 Sentence denotes In conclusion, the visual LAMP assay developed here for detecting ZIKV is more sensitive than PCR and RT-PCR and the results can be observed by naked eyes immediately when amplification is completed.
T111 16292-16414 Sentence denotes For sensitive and specificity, the results of this visual method were consistent with real-time turbidimeter observations.
T112 16415-16536 Sentence denotes Consequently, a specific, sensitive, rapid, and cost-effective means for ZIKV detection with LAMP method was established.
T113 16537-16623 Sentence denotes It is very helpful for primary health care units to use the LAMP assay to detect ZIKV.