Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip

Abstract

We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.

Fig.1

Principle of multisample detection DNA chip. In the LAMP method, six primer regions are set and four primers are used for amplification. The F3, F2, and F1 regions are placed in this order from the 5′ terminal side of a forward strand of a target nucleic acid, and the B3c, B2c, and B1c regions are placed in this order from the 3′ terminal side of the forward strand of a target nucleic acid. The complementary regions of F3, F2, F1, B3c, B2c, and B1c in the reverse strand are called F3c, F2c, F1c, B3, B2, and B1 regions, respectively. Primers constituting four basic primers are FIP (F1c + F2), BIP (B1c + B2), F3, and B3. In addition, the amplification period can be shortened by optionally using a primer called a loop primer, LPc. FIP primer hybridizes to F2c in the target nucleic acid and initiates complementary strand synthesis, and then F3 primer hybridizes to F3c in the target nucleic acid and initiates strand displacement DNA synthesis, releasing an FIP-linked complementary strand. When F1c and F1 that exist in the FIP-linked complementary strand bind, the F2 region and F2–F1 region become a single-stranded loop. Similarly, the B2 region and B2–B1 region become a single-stranded loop. In this multisample detection DNA chip, first, the primers that insert a different tag sequence in each sample are prepared. Second, the amplification reactions are performed independently in each sample using these primers. In samples 1 and 3 where the target nucleic acid exists, when the tag insertion primer binds to the target nucleic acid, the tag part causes the loop out and the amplification reaction can proceed. Then amplification products in which a part of the sequence was different in each sample can be obtained. Finally, the target sample in which the amplification products from each sample are mixed is detected by the DNA chip.

Fig.2

Position of LAMP primer and target sequence. The sequence alignment of the 16S rRNA gene of Helicobacter species is shown. The numbers after species names are GeneBank accession numbers. The arrows, bold-line squares, and triangles indicate the LAMP primer positions, target sequence positions, and tag insertion positions, respectively. Tag sequences that inserted LAMP primers are listed in Table 1. The dotted-line squares indicate the specific sequence regions between Helicobacter species.

Fig.3

Detection results of five tag introduction products by Genelyzer. 1.CTG is the detection result of a target sample containing a positive product from CTG tag insertion primer and negative products from GGA, CCT, TCC, and ATC tag insertion primers. Similarly, 2.GGA, 3.CCT, 4.TCC, and 5.ATC are the detection results of target samples containing one positive product and four negative products. Here 5 μl of each product was used for detection.