Method for detecting a plurality of short-chain nucleic acid in sample, combinatorial analysis kit, analysis kit supply management method

Abstract

According to one embodiment, a detection method is a method for detecting a plurality of target nucleic acids in a sample. The method includes (a) preparing a chain-elongation nucleic acid set group, a primer set, and a probe immobilized substrate, (b) obtaining the target nucleic acid and a long-chain nucleic acid group containing a first sub-chain-elongation nucleic acid and a second sub-chain-elongation nucleic acid, (c) obtaining an amplification product group by maintaining the long-chain nucleic acid group and the primer set under amplification conditions, (d) detecting presence/absence and/or an amount of hybridization, and (e) detecting the plurality of target nucleic acids.

1 - 16 . (canceled) 
     
     
         17 . A combinatorial analysis kit for detecting a plurality of target nucleic acids of short chain, wherein
 the kit includes   (1) a reagent that elongates each of the plurality of target nucleic acids containing a plurality of target sequences having different sequences from each other, respectively and forms a plurality of long-chain nucleic acids to obtain amplification products of the long-chain nucleic acids and   (2) at least one probe immobilized substrate provided in combination with the reagent configured to perform amplification of the plurality of long-chain nucleic acids and detection of the amplification products, wherein   each of the plurality of target nucleic acids is a short-chain nucleic acid having a first sub-target sequence at a 5′-terminal and a second sub-target sequence at a 3′-terminal,   (1-1) the reagent contains   (a) a plurality of chain-elongation nucleic acid groups containing a first sub-chain-elongation nucleic acid and a second sub-chain-elongation nucleic acid corresponding to each of the plurality of target nucleic acids,   wherein   the first sub-chain-elongation nucleic acid contains a first target binding region containing the first sub-target sequence or a first complementary sequence complementary thereto at one terminal and a first amplification region at another terminal and   the second sub-chain-elongation nucleic acid contains a second target binding region containing the second sub-target sequence or a second complementary sequence complementary thereto at the 3′-terminal and a second amplification region at the 5′-terminal and   wherein, at least one of the first sub-chain-elongation nucleic acid and the second sub-chain-elongation nucleic acid is further associated with each of the plurality of target sequences in advance and contains one corresponding detection region among a plurality of detection regions having different sequences from each other,   when the first sub-chain-elongation nucleic acid contains a first detection region, the first detection region is present between the first target binding region and the first amplification region without overlapping the first target binding region and,   when the second sub-chain-elongation nucleic acid contains a second detection region, the second detection region being present between the second target binding region and the second amplification region without overlapping the second target binding region and   (b) a universal primer set containing at least a first primer containing a sequence common among the plurality of target nucleic acids and bound to the first amplification region, and a second primer containing a sequence common among the plurality of target nucleic acids and bound to the second amplification region, and   (2-1) the probe immobilized substrate includes   a substrate and   a plurality of probes immobilized to a surface in contact with a first reaction field provided by the substrate,   wherein each of the plurality of probes   (1) contains a same sequence as that of a respective one of the plurality of sequences of the first detection region,   (2) contains a complementary sequence to a respective one of the plurality of sequences of the first detection region,   (3) contains a same sequence as that of a respective one of the plurality of sequences of the second detection region, and/or   (4) contains a complementary sequence to a respective one of the plurality of sequences of the second detection region.   
     
     
         18 . The kit of  claim 17 , wherein the universal primer set is one intended for PCR,
 a combination of the first primer and the second primer is a combination of a forward primer and a reverse primer,   the plurality of target nucleic acids are first to m-th target nucleic acids, where m is an integer equal to 2 or greater,   the plurality of target sequences are first to m-th target sequences corresponding respectively to the target nucleic acids, the first sub-target sequences are 1 1  to 1 m -th sub-target sequences corresponding respectively to the plurality of target sequences, the second sub-target sequences are 2 1  to 2 m -th sub-target sequences corresponding to respectively the plurality of target sequences,   the sequence of the first amplification region is common among 1 1  to 1 m -th sub-chain-elongation nucleic acids, the first amplification region contains a sequence that binds to the sequence of the first primer,   the sequence of the second amplification region is common among 2 1  to 2 m -th sub-chain-elongation nucleic acids, and the second amplification region contains a sequence that binds to the sequence of the second primer.   
     
     
         19 . The kit of  claim 17 , wherein amplification conditions are reaction conditions of LAMP,
 the target nucleic acids are first to m-th target nucleic acids, where m is an integer equal to 2 or greater,   the plurality of target sequences are first to m-th target sequences corresponding to each of the plurality of target nucleic acids, the first sub-target sequences are 1 1  to 1 m -th sub-target sequences corresponding to each of the plurality of target sequences, the second sub-target sequences are 2 1  to 2 m -th sub-target sequences corresponding to each of the target sequences,   the first amplification region is an F2 binding region, and the first sub-chain-elongation nucleic acid contains an F1 binding region, the F1 binding region being present between the F2 binding region and a first sub-target binding region,   when the first detection region is contained in the first sub-chain-elongation nucleic acid, the first detection region is present, on the first sub-chain-elongation nucleic acid, between a base adjacent to the F1 binding region and a region overlapping with the F2 binding region to avoid overlapping with the F1 binding region, and contains a region without overlapping the F2 binding region,   the sequence of the second amplification region is a B2 binding region, and the second sub-chain-elongation nucleic acid contains a B1 binding region, the B1 binding region being present between the B2 binding region and a second sub-target binding region,   when the second detection region is contained in the second sub-chain-elongation nucleic acid, the second detection region is present, on the second sub-chain-elongation nucleic acid, in a range on the 3′-terminal side from the 5′-terminal of the B2 binding region from a 5′-terminal side bases at the 5′-terminal of the B1 binding region,   the first primer is an FIP primer containing an F1c sequence at the 5′-terminal and an F2 sequence at the 3′-terminal,   the second primer is a BIP primer containing a B1c sequence at the 5′-terminal and a B2 sequence at the 3′-terminal,   the first detection regions are 1 1  to 1 m -th detection regions corresponding respectively to the target sequences, the second detection regions are 2 1  to 2 m -th detection regions corresponding respectively to the target sequences, and   the probes are 1 1  to 1 m -th probes and/or 2 1  to 2 m -th probes corresponding respectively to the first detection region and/or the second detection region.   
     
     
         20 . The combinatorial analysis kit of  claim 17 , wherein
 the reagent in the above (1) is a first reagent associated with a first series and the plurality of target sequences for the first reagent is a target nucleic acid group associated with the first series and   the combinatorial analysis kit further comprises:   (3) at least one of second to n-th reagents associated with second to n-th series respectively provided in combination with the first reagent in the above (1),   wherein   each of the second to n-th reagents is associated with each of the second to n-th series and is a reagent that elongates each of the plurality of target nucleic acids containing the plurality of target sequences mutually different in the series respectively, forms the plurality of long-chain nucleic acids, and obtains amplification products of the long-chain nucleic acids; and/or   (4) probe immobilized substrate provided in combination with at least one of the above (1) and/or the second to n-th reagents in the above (3),   wherein   first to n-th small item sets containing m 1  to m n  small items preset for each of the first to n-th series are set respectively, numbers of the small items contained in the first to n-th small item sets are m 1  to m n  respectively, m 1  to m n  are identical to each other or different from each other, a maximum number in the first to n-th small item sets is m max , m and n are independent of each other and integers equal to 2 or greater, and types the small items contained in each of the first to n-th series are at least partially between the series,   any x-th series of the first to n-th series is analyzed using a corresponding x-th small item set and a x-th small item set contains 1 x -th to m x -th small items and 1≤m x ≤m max  applies,   the 1 x -th to m x -th small items are intended to obtain presence or an amount of presence of each of 1 x -th to m x -th target nucleic acids having 1 x -th to m x -th target sequences having different sequences from each other, respectively and the 1 x -th to m x -th target sequences contained in the x-th small item set are a target nucleic acid group related to a theme common throughout the x-th series, and   the 1 x -th to m x -th target sequences contain 1 1x -th to 1 mx -th sub-target sequences at the 5′-terminal and 2 1x -th to 2 mx -th sub-target sequences at the 3′-terminal respectively.   
     
     
         21 . A supply management method of the combinatorial analysis kit of  claim 17 , comprising:
 constructing a database that stores inventory information associated with a location having the kit and relating to chain-elongation nucleic acid sets and the probe immobilized substrate independently of each other; and   searching for the inventory information in the database by a management system having received an order of the kit from a user in accordance with content of the order, determining the location having the kit, the chain-elongation nucleic acid set, or the probe immobilized substrate as a stock to be sent to the user according to preset conditions from information contained therein, and issuing instructions to send from the location to the user.   
     
     
         22 . The kit of  claim 17 , wherein the kit comprises at least one of the probe immobilized substrate configured to perform an amplification reaction of a plurality of types of elongated products and detection of amplification products obtained by the amplification reaction in a same reaction field in a same period. 
     
     
         23 . A probe immobilized substrate configured to perform an amplification reaction of a plurality of types of elongated products obtained by elongating a plurality of types of target nucleic acids, which are short-chain nucleic acids, in a sample using a plurality of types of chain-elongation nucleic acid sets and detection of amplification products obtained by the amplification reaction in a same reaction field, wherein the probe immobilized substrate comprises:
 a substrate,   a plurality of probes immobilized to a surface in contact with a first reaction field provided by the substrate, and   a single type of universal primer set for amplifying a plurality of the elongated nucleic acids, releasably immobilized to a surface in contact with the first reaction field,   wherein each of the plurality of probes   (1) contains a same sequence as that of a respective one of the plurality of sequences of the first detection region,   (2) contains a complementary sequence to a respective one of the plurality of sequences of the first detection region,   (3) contains a same sequence as that of a respective one of the plurality of sequences of the second detection region, and/or   (4) contains a complementary sequence to a respective one of the plurality of sequences of the second detection region.   
     
     
         24 . The probe immobilized substrate of  claim 23 , wherein
 the first reaction field is a channel, and   the universal primer set is releasably immobilized to a surface in contact with a first reaction field which is located at a upstream end of the channel.