TWI460275B - Oligonucleotide biochip and methodology for identification of crucial antrodia and antrodiella species - Google Patents

Description

Oligonucleotide biochip for identifying Antrodia and Antrodiella and method thereof

The present invention relates to an oligonucleotide biochip capable of identifying a genus of the genus Phaeoporus and the genus Phaeoporococcus, and a method for identifying the genus Phaeoporus and the genus Phaeoporococcus using the wafer.

The identification of traditional fungi is based on the characteristics of microscopic or macroscopic forms, structure and sporegenesis of fungi, but it is necessary to correctly identify these fungi with various forms, structures, structures and high degrees of heterogeneity. Learning, education, training, practical operations and experience learning. In recent years, the rapid progress of molecular biology has made significant classification progress and breakthroughs in species identification and taxonomy and phylogeny, such as amplified fragment length polymorphism (AFLP). Random amplification of polymorphic DNA (DNA), simple sequence repeat (SSR), sequence characterized amplified region (SCAR) , nano-magnetic bead polymerase chain reaction (NMB-PCR), multi-plex PCR, microsphere-based Array, etc. (Krokene et al . 2004;. Wang et al 2009 ;. Hopfer et al 1993;. Rath et al 2000;. Guglielmo et al 2008;. Lin et al 2008.), but there is no funding can be applied in a thin layer of rapid diagnostic species ( Antrodia ) and fungi that are close to each other. Therefore, it provides a convenient way for rapid and accurate diagnosis, detection and identification of plant pathogens and medicinal fungi, and needs it.

The nucleic acid of all biological species has a so-called highly repetitive sequence of ribosomal RNA (rRNA), which is mainly composed of 18S-ITS1-5.8S-ITS2-28S. ITS1 and ITS2 are called internal transcribed spacers. This sequence can be spliced when ribosomal DNA (rDNA) transcribes rRNA (mRNA), so it has no function, also known as pseudointron. (pseudointron), the variability is higher, and the adjacent 5.8S, 18S and 28S rRNA have translation function, which is more conservative, so the variability is lower; due to the dual characteristics of rRNA (rDNA) with variability and conservation, Therefore, it can be used for more or less sequence differences, and it is an objective reference standard for the study of intra-species, interspecies, intergeneric and even genus classifications and molecular kinship. This also means that the difference in rDNA sequences can be applied. Tools such as reverse blot hybridization or microarray to identify similar species. The ITS1-5.8S-ITS2 of rDNA is about 500-900 base pairs in length, and its species has a similarity of about 40-99%, and each species often has 140 copies, which are beneficial. It is designed as a specific probe for diagnosis and identification.

Antrodia (Antrodia) under Basidiomycotina (Basidiomycotina), as Polyporus (polypores), fruiting bodies of a flat type (resupinate), spores or teardrop-shaped and cylindrical, thin-walled smooth, non-coupling bacteria Silk (binding hyohae). The genus Phaeoporus was first established in 1880 by Karsten. Later, it was revised several times by Ryvarden to form a more stable taxonomic group (Gilbertson and Ryvarden, 1986). He narrowed the definition of the genus, focusing on the characteristics of wood decay, ecology, hyphae and spores. Some of the genus Pseudomonas are saprophytic, some are weakly pathogenic, often causing brown rot and root rot in the roots of the trees. If the habitat is poor, it is more likely to cause the tree to fade and yellow, and the trees are vulnerable to wind and wind. Common of this species contains Antrodia albida (A. albida), white brown thin volvatus (A. albobrunnea), thin mountain volvatus (A. alpina), a carbon thin volvatus (A. carbonica), Antrodia cinnamomea (A . cinnamonea ), A. gossypina , A. juniperina , A. lalashana , A. malicola , fragrant thin hole A. odora , A. oleracea , A. radiculosa , A. salmonea , A. serialis , wavy thin A. sinuosa , A. sitchensis , A. sordida , A. taxa , A. vaillantii And A. xantha . Among them, A. cinnamomea ( A. sylvestris ) and A. salmonea ( Cymbidium sinensis ) are endemic species of Taiwan, and A. angustifolia is often regarded as an auxiliary medicinal material in the folk because of its physiologically active substances.

A. cinnamomea is endemic to Taiwan and belongs to Polyporaceae. In 1995, Zhang Dongzhu and Zhou Wenneng made detailed descriptions on the appearance, color, odor, growth rate and spore microstructure of Antrodia camphorata. Name it " Antrodia cinnamomea " TT Chang & W. N. Chou (Chang and Chou, 1995). It grows on the inner wall of the burdock cavity and is slow, causing brown rot of the heart of the burdock. The fruiting body has a wide and tight contact area with wood, and has a plate shape or a bell shape, has no handle, and has a pale yellow-white cork on the bottom layer, whereby the cork is attached and grown on the inner wall of the hollow material. In the shape of a bell, the fruit layer is orange-red, the pores are orange-brown to pale cinnamon, the pores are fine and dense, 4-5 per millimeter, and the appearance is round to angular, lined with a load plate and a spore. Basidiospore is oval, smooth, and thin-walled (Zhang et al., 1999). Nigella sinensis causes browning of heartwood, and decay will reduce the texture and volume of burdock wood. However, in recent years, Antrodia camphorata has found a variety of physiologically active substances in its research. It has been reported to have anti-tumor, enhance immunity, protect liver, and resist hypertension. Reduce blood sugar, anti-bacterial, anti-oxidation, reduce cardiovascular diseases and other functions (Geethangili and Tzeng, 2009).

The appearance, ecological and even medicinal effects of the fruiting body of A. salmonea is very similar to that of A. sylvestris , but it is parasitic on fragrant fir wood, mainly due to the decay of wood at the root of the stem. However, there is still a slight difference between Xiangshanzhi and Antrodia camphorata. The fresh fruiting body of Xiangshanzhi is pale pink to pale yellow, while the aconite is dark reddish brown. The habitat distribution of the two is different. The distribution of Antrodia camphorata is about 1000. -1600 meters, while the cedar is grown in the mountains of about 1500-3500 meters. In addition, the burdock has a rich scent, and the cedar is the scent of the fir itself, which can be slightly different.

Antrodiella belongs to the genus Basidiomycotina and belongs to the flat polyporous bacterium. It is very similar to the microscopic characteristics of the genus Pseudomonas. For example, the hyphae is a dimitic hyphal system: skeletal hyphae (skeletal hyphase) is a transparent thick wall, generative hyphase containing clamps; no vesicles, no spores, no starch and cellulose (Ryvarden, 1991). It is distinguished from the form of decay of wood caused by the genus Phaeoporus. The former is white rot, which can secrete wood, fiber, and semi-fibrinolytic enzymes, which can decompose cellulose and wood. The quality of the remaining rot wood is white; the latter is brown rot, secreting fiber or semi-fibrous enzyme, but the salt letter does not have the ability to decompose lignin, and the decomposed wood leaves brown lignin and is called. The important pathogens of this genus include A. americana , Taiwan's A. formosana , A. liebmannii , and A. romellii . It is half-like like A. semisupina .

Due to the chronic atrophy of forest trees caused by these forest pathogenic fungi, causing poor growth, weakening, defoliation, decay, and lodging, it has become a major limiting factor and a potential serious threat to the forestry industry. In recent years, research on Antrodia camphora has found that it has a variety of medical physiological functions. More and more people in the industry expect to develop artificial cultivation to mass produce fruiting bodies, but it is not easy. To ensure the correctness of the provenance, wafers can be used. filter. In addition, the collection of wild Antrodia camphorata is not easy and illegal. Although it is artificially cultivated, it seems to have not been popularized. Therefore, the fruiting body is expensive, and the unscrupulous people often use the similar appearance of Pycnoporus sanguineus and A. salmonea to counterfeit. The fish are mixed with beads to deceive consumers, and many manufacturers use the scorpion smashed into fine powder to fill the capsules, but the ingredients are really difficult to distinguish, and the job is the reason. If the biochip can be developed, it can be used in a very short time. To accurately detect, identify and distinguish between authenticity and falsehood to protect the rights and interests of consumers.

The present invention discloses a biochip which can be used for identifying P. ycnoporus sanguineus , Antrodia and Antrodiella , on which a plurality of probes are immobilized. Wherein the plurality of probes are selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5. SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20 oligonucleotide sequences, complementary strands thereof, derivatives And combinations thereof, wherein the derivative refers to an oligonucleotide which modifies other nucleotide sequences at the 3' or 5' end of the sequence or its complementary strand so that it still has 70%-100% similarity to the original sequence. sequence. The biochip can be used for the identification of forest diseases caused by the genus Pseudomonas and the like, and the afforestation management, the health seedling certification and the detection and identification of the product of the market.

The present invention also discloses an oligonucleotide sequence for identifying Pycnoporus sanguineus , which is the sequence shown in SEQ ID No. 1, a complementary strand or a derivative thereof, wherein the derivative refers to the sequence or The 3' or 5' end of the complementary strand modifies the other nucleotide sequence such that it still has 70%-100% similarity to the original sequence.

The invention further discloses an oligonucleotide sequence for identifying an genus Antrodiella , comprising: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5 A sequence, a complementary strand or derivative thereof, wherein the derivative refers to a modification of the other nucleotide sequence at the 3' or 5' end of the sequence or its complementary strand, such that it remains 70%-100% identical to the original sequence. Sexual oligonucleotide sequence.

The invention also discloses an oligonucleotide sequence for identifying Antrodia , comprising: SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. .18, the sequence of SEQ ID No. 19 or SEQ ID No. 20, a complementary strand or derivative thereof, wherein the derivative refers to the modification of other nucleotides at the 3' or 5' end of the sequence or its complementary strand The sequence is such that it remains 70%-100% similar to the original sequence.

The invention further discloses a method for identifying Pycnoporus sanguineus , Antrodia and Antrodiella , comprising the steps of: (a) extracting deoxyribose from a strain to be tested; Nucleic acid (DNA); (b) extracting the ITS1-5.8S-ITS2 ribosomal DNA (rDNA) fragment sequence in the DNA by an appropriate primer pair; (c) the product of step (b) and the invention The biochip is subjected to a hybridization reaction; and (d) determining the genetic species of the test strain by observing which product of the step (b) has a positive reaction with a probe on the biochip. Preferably, in this method, a primer pair consisting of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22 is used, and the amplification is carried out by a polymerase chain reaction. In the identification of the strain, if the product of the step (b) has a positive reaction with the SEQ ID No. 1 oligonucleotide sequence, its complementary strand or derivative in the biochip described in claim 1 of the patent application. , the strain to be tested is identified as Pycnoporus sanguineus ; the product of step (b) is the SEQ ID No. 2 oligonucleotide sequence in the biochip described in claim 1 If the complementary strand or derivative has a positive reaction, the strain to be tested is identified as Antrodiella americana Ryvarden et Gilbertson; the product of step (b) and the biochip described in claim 1 The SEQ ID No. 3 oligonucleotide sequence, its complementary strand or derivative has a positive reaction, and the strain to be tested is identified as a product of the order of the Taiwanese microorganism ( Antrodiella formosana Chang et Chou; step (b) In the case of a positive reaction with the SEQ ID No. 4 oligonucleotide sequence, its complementary strand or derivative in the biochip described in claim 1, the strain to be tested is identified as a tough-like fungus ( Antrodiella liebmannii (Fries) Ryvarden); the production of step (b) If the strain has a positive reaction with the SEQ ID No. 5 oligonucleotide sequence, the complementary strand or the derivative thereof in the biochip described in claim 1, the strain to be tested is identified as a semi-negative thin hole. Antrodiella semisupina (Berkeley et Curtis) Ryvarden; the product of step (b) and the SEQ ID No. 6 oligonucleotide sequence, complementary strand or derivative thereof in the biochip of claim 1 If there is a positive reaction, the strain to be tested is identified as Antrodia albida (Fries) Donk; the product of step (b) is SEQ ID No. in the biochip described in claim 1 of the patent application. 7 The oligonucleotide sequence, its complementary strand or derivative has a positive reaction, then the strain to be tested is identified as Antrodia albobrunnea (Romell) Ryvarden; the product line of step (b) and the scope of patent application The SEQ ID No. 8 oligonucleotide sequence in the biochip of item 1, the complementary strand or derivative thereof has a positive reaction, and the strain to be tested is identified as Antrodia alpina (Litschauer) Gilbertson et Ryvarden); product of step (b) and patent application scope 1 The SEQ ID No. 9 oligonucleotide sequence, its complementary strand or derivative in the biochip described has a positive reaction, and the strain to be tested is identified as Antrodia carbonica (Overholts) Ryvarden et al .); the product of step (b) is positively reacted with the SEQ ID No. 10 oligonucleotide sequence, its complementary strand or derivative in the biochip of claim 1 of the patent application, and the test is to be tested The strain is identified as Antrodia cinnamomea Chang et Chou; the product of step (b) is the SEQ ID No. 11 oligonucleotide sequence, its complementary strand or derivative in the biochip described in claim 1 With a positive reaction, the strain to be tested is identified as Antrodia juniperina (Murrill) Niemel Et Ryvarden); the product of step (b) is positively reacted with the SEQ ID No. 12 oligonucleotide sequence, its complementary strand or derivative in the biochip of claim 1 of the patent application, The strain was identified as TFRI 550 ( Antrodia lalashana Chang et Chou (TFRI 550)); the product of step (b) and SEQ ID No. 13 in the biochip described in claim 1 If the oligonucleotide sequence, its complementary strand or derivative has a positive reaction, the strain to be tested is identified as TFRI 1607 ( Antrodia lalashana Chang et Chou (TFRI 1607)); the product of step (b) Is a positive reaction with the SEQ ID No. 14 oligonucleotide sequence, its complementary strand or derivative in the biochip described in claim 1 of the patent application, and the strain to be tested is identified as an apple bacterium ( Antrodia) Malicola (Berkeley et Curtis) Donk); the product of step (b) has a positive reaction with the SEQ ID No. 15 oligonucleotide sequence, its complementary strand or derivative in the biochip of claim 1 , the strain to be tested is identified as Antrodia oleracea (Davidson et Lo [ mbard) Ryvarden); the product of step (b) is positively reacted with the SEQ ID No. 16 oligonucleotide sequence, its complementary strand or derivative in the biochip of claim 1 The strain to be tested is identified as Antrodia salmonea Chang et Chou; the product of step (b) is complementary to the oligonucleotide sequence of SEQ ID No. 17 in the biochip described in claim 1 If the strand or derivative has a positive reaction, the strain to be tested is identified as Antrodia serialis (Fries: Fries) Donk; the product of step (b) and the organism described in claim 1 The SEQ ID No. 18 oligonucleotide sequence in the wafer, its complementary strand or derivative has a positive reaction, and the strain to be tested is identified as Antrodia sordida Ryvarden et Gilbertson; the product of step (b) And if the SEQ ID No. 19 oligonucleotide sequence, the complementary strand or the derivative thereof in the biochip described in claim 1 has a positive reaction, the strain to be tested is identified as a porcine porcine ( Antrodia taxa Chang et Chou); or the product of step (b) and the scope of patent application 1 The SEQ ID No. 20 oligonucleotide sequence, its complementary strand or derivative in the biochip described has a positive reaction, and the strain to be tested is identified as Antrodia xantha (Fries) Ryvarden.

The embodiments of the present invention will be further described in conjunction with the accompanying drawings. The embodiments of the present invention are not intended to limit the scope of the present invention, and those skilled in the art, without departing from the spirit of the invention. In the scope of the invention, the scope of the invention is defined by the scope of the appended claims.

The invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the various aspects and features of the present invention. The described embodiments are not intended to limit the scope of the invention as described in the claims.

Example 1 Oligonucleotide Construction Method and Process (1) Strains and culture Source of strain

The test strains for this study were from the Taiwan Forestry Laboratory, the Bioresource Collection and Research Centre (BCRC, Biossource, Research Center, Hsinchu, Taiwan, ROC) and the US Department of Agriculture, Wisconsin New Territories. Forest Fungus Research Center (USDA Forest Product Laboratory. Wisconsin, USA) (Table 1).

bacteria Body culture

The obtained strain was cultured in a plate or a beveled test tube made of potato dextrose agar (Difico, USA) at 25 ° C for 7-10 days for the source of the subsequent test.

(2) Extraction of bacterial DNA Small amount DNA extraction of cetyltrimethylammonium bromide (CTAB)

The hyphae were scraped from the plate medium, and about 0.1 g was placed in a microcentrifuge tube, and 500 μl of CTAB buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, and 100 mM Tris (pH 8), 2% 65 ° C preheated was added. PVP-40), grind to a rice flour using a tissue grinding rod, add 3 μl of 2-mercaptoethanol, mix well by shaking, place at 65 ° C for 10-20 minutes, then add 500 μl CI (chloroform: Isoamyl alcohol = 24:1), gently mix up and down, then centrifuge at 13,200 rpm for 2 minutes, remove the supernatant (about 500 μl) into a new microcentrifuge tube, add 300 μl (0.6 volumes) of isopropyl The alcohol was slightly spun up and down to mix the DNA, followed by centrifugation at 13,200 rpm for 2 minutes, carefully removing the supernatant, adding 500 μl of wash buffer (76% ethanol, 10 mM ammonium acetate), gently shaking and placing 2 After centrifugation at 13,200 rpm for 2 minutes, the supernatant was removed, dried by centrifugation in a Labconco vacuum dryer, and dissolved by adding 200 μl of ddH ₂ O.

Extraction of DNA from forest rickets tissue or fruiting body

The rickets to be tested are scraped into small pieces and added with liquid nitrogen. The tissue is ground into a powder using a tissue grinding rod, and about 0.1 g of the powder is placed in a microcentrifuge tube. The subsequent small amount of DNA extraction is the same as above.

(3) Increase in the transcribed region (ITS) of DNA (PCR reaction) Primers and conditions for PCR use

The chromosomal nucleic acid of the fungus was extracted by the method reported by Al-Samarrai and Schmid (2000), and the obtained chromosomal nucleic acid was stored at -20 ° C for use. The increase and sequence of the internal transcribed spacer (ITS) gene sequence using the primer designed by White et al. (1990) to amplify the ITS1-5.8S-ITS2 rDNA fragment sequence, the primer sequence used is ITS4e ( SEQ ID No. 21, 5'-TCCTCCGCTTATTGATATGCTTAAG-3') and ITS5e (SEQ ID No. 22, 5'-TTAGAGGAAGTAAAAGTCGTAACAAGGTT-3'). The PCR procedure is as follows: 50 μl reaction volume (35.5 μl ddH ₂ O, 5 μl 10 times reaction concentration buffer, 10 mM dNTP 1 μl, 20 μM ITS4e primer 1.25 μl, 20 μM ITS5e primer 1.25 μl, template DNA 5 μl , 1 unit taq polymerase) was placed in a polymerase chain reaction thermal reactor (Biometra T3 thermocycler) for amplification. The temperature reaction conditions were 94 ° C, 4 minutes later; 94 ° C, 1 minute, 50 ° C, 1 minute, 72 ° C, 1 minute, three temperature cycles 35 times; then 72 ° C, 7 minutes. After the PCR reaction, electrophoresis analysis was carried out with 1% acacia to determine the presence or absence of PCR nucleic acid amplification products.

PCR product purification

The PCR product was purified by GFXTM PCR DNA Purification Kit (Amersham Pharma), first electrophoresed in 1.5% agar colloid, electrophoresis at a voltage of 100 V for 25 minutes, and each well can be injected with 13-14 μl DNA sample to reduce The amount of colloid should be cut. The film was stained with ethidium bromide (Ethidium Bromide) for 5 minutes and placed in a MODEL TL-312A Transilluminator 312 nm Ultraviolet ( ), cut the desired fragment. The cut colloid was placed in a microcentrifuge tube, and the capture buffer of the GFXTM PCR DNA purification kit was added. After that, the mixture was shaken uniformly and placed in a 60 ° C dry bath for 10 min. Centrifuge briefly at room temperature and assemble the GFX column into a collection tube. Next, the sol-containing liquid was transferred to a GFX column and placed at room temperature for 2 minutes and then centrifuged at 13,200 rpm for 30 seconds. The GFX column was removed and the liquid in the collection tube was removed. The GFX column was placed on a collection tube and 500 μl of wash buffer was added and centrifuged at 13,200 rpm for 30 seconds. The liquid in the collection tube was removed and the GFX tube was placed back on the collection tube and centrifuged at 13,200 rpm for 2 minutes. Then, 50 μl of TE buffer was added to the center of the bottom of the column, and after standing at room temperature for 2 minutes, it was centrifuged at 13,200 rpm for 2 minutes to collect the centrate.

(4) Selection and sequencing of ITS fragments of different forest pathogenic fungi

Since the product obtained by PCR with Taq DNA polymerase has a common characteristic, that is, an adenine is added at the 3' end position, a thymine is added to the 5' end of the vector, and DNA ligase is used. And adenosine triphosphate (ATP) work together to cloning the PCR product and the carrier on the principle of TA adhesion. The system used in this experiment is pGEM -T Easy Vector system (Promega).

Bond the product to pGEM -T Easy Vector

The reaction mixture was prepared as follows: 10X T4 DNA ligase ligation buffer 1 μl, 50 ng pGEM -T Easy Vector 1 μl, purified PCR product 7 μl, T4 DNA ligase (3 Weiss Units/μl) 1 μl, the mixture was placed in a microcentrifuge tube and allowed to act overnight at 4 °C. In this experiment, TA selection was applied to the selection of specific probe fragments for probe preparation and preservation.

E . Coli DH5α Transformation

In order to prepare and store a large amount of the plastid containing the probe fragment, the competent cells made of E. coli DH5α are transformed, and the plastid containing the probe fragment is sent to the competent cells. 100 μl of HITTM DH5α competent cells (RBC, Taiwan) stored at -80 ° C were taken and placed on ice to be thawed. The junction product was briefly centrifuged, 4 μl was added to the competent cells, gently stirred with a micropipette to make it evenly mixed, and the microcentrifuge tube was placed on ice for 20 minutes, then in a water bath at 42 ° C for 45-50 seconds, rapidly After the tube was placed on ice for 2 minutes, 900 μl of LB medium was added, and cultured at 37 ° C with shaking at 150 rpm for 1-2 hours. Then, 80-100 μl of the L-shaped glass rod was uniformly applied to the LB/Ampicillin/X-Gal plate medium, and the mixture was repeated for 14-16 hours at 37 ° C. Next, a single white colony was picked with a 200 μl micropipette tip and inoculated on two LB plate medium containing 100 μg/ml Ampicillin (the PCR method can be directly used to remove the residual bacteria on the micropipette nozzle). Initial confirmation of colony).

PCR product sequencing

The colonies that were successfully selected were sent to the biotechnology company for sequencing, and the automated nucleic acid sequencer (Applied Biosystems, ABI 3730, Taipei, Taiwan) was used for sequencing, and the BigDye Terminators were used for fluorescent labeling. The photometer can be interpreted with the computer automatically to interpret 1kb, and its accuracy is over 98.5%.

Sequence analysis

The sequence sequence was corrected using the ContigExpress function in the computer software Vector NTI 9.0 (Infor Max Inc., USA), and the alignment of the nucleic acid sequences was performed by AlignX to search for specific fragment design probes in the sequence. After the comparison results appear, select Align→show similarity table on the toolbar to get the similarity of ITS between species.

( 5) Probe design (Probe design)

Sequences obtained by sequencing the strains were aligned with the ITS sequences using Vector NTI 9.0 (Invitrogen, CA, USA) software, and the specific design probes between the different strains were found. The probe design principles are as follows: (1) each probe is about 25-60 nucleotides in length; (2) the double-stranded nucleic acid dissociation temperature (Tm value) of each probe needs to be between 60-85 °C. (3) Add 14 T (thymine) to the 3' end of the short probe (less than 40 bases), and add 3 Ts to the 3' end of the long probe (greater than 40 bases) to enhance the signal response; (4) The GC content is about 40-60%, the ratio should not be too high to reduce the non-specific heterozygous reaction; (5) avoid selecting the sequence and secondary structure of multiple consecutive identical bases (for example, 5 consecutive C) (secondary structure) (eg, dimer, hairpin structure, etc.); (6) try to design the position of the differential base in the middle of the probe. After all probes were designed, they were aligned with the sequences on GenBank using BLAST to check whether they would cross-react with other strains. The probe is named after the first two letters of the generic name with the first three letters of the species name. Antrodia cinnamomea is taken as an example. The probe is named Ancin, and all probe sequence data are listed in Table 2. The probe was commissioned by Tri-I biotech Inc. (Taipei, Taiwan).

( VI) Preparation and use of wafers Probe preparation

Prepare a 96-well round bottom ELISA plate, dissolve the probe in water, with a tracking dye [30% (v/v) glycerol, 1 mM disodium edetate (EDTA), 40% ( v/v) dimethyl hydrazine, 0.15% (w/v) bromophenol blue, 10 mM Tris-HCl, pH 7.5] mixed in a ratio of 1:1 (v/v), the final concentration of the probe is divided by the coordinate probe In addition, all are 20 μM, while the coordinate probe is labeled with digoxigenin (DIG) or biotin (biotin) at the 5' end of oligo-(dT) ₁₀ at a concentration of 1 μM. EzspotTM Arrayer SR-A300 (Yuesheng Technology, Taipei, Taiwan), using a solid pin of 400 μm diameter for wafer spotting, each sample was probed to a nylon membrane (positively charged, Nylon membrane, Roche) On Mannheim, Germany, the center distance between the points is 800 μm. After the wafer was spotted, it was air-dried at room temperature, and then irradiated with 1.2 J of ultraviolet light (Stratagene, USA), and the probe was fixed on a nylon membrane and stored in a dark dry place for use. The probe configuration of the Antrodia chip is shown in Figure 2.

Preparation of target DNA

The primer pair (ITS4e and ITS5e) used in the amplification of the above internal transcribed spacer (ITS) gene sequence was replaced with a primer pair of biotin (Biotin) at the 5' end (MDbio, Inc., Neihu, Taiwan), the method and process of PCR are the same as above.

Preparation of hybrid control sample (Hybridization control sample)

The heterozygous control group was designed as a hybrid control sample using the sequence of the KS region of the fungal black synthesis-related gene (polyketide synthase), first using the primer pair CoKS1 (SEQ ID No. 23, 5'-TTCAACATGTCTCCCCGTGAGG-3'), CoKS2 (SEQ ID No. 24, 5'-AAGGGGGTCGACACCAGCATCGTCG-3') PCR amplification of the DNA of Colletotrichum graminicola BCRC 35078, the amplification conditions are the same as the aforementioned ITS increase, The amplified product is sequenced and compared according to the above-mentioned gene selection method, and the probe is designed as a probe of the heterozygous control group according to the optimal condition on the search sequence (according to the above probe design principle), and will be selected. After the plasmid was extracted, the sequence was diluted, and the plastid primer paired T7 (SEQ ID No. 25, 5'-TAATACGACTCACTATAGGG-3') and SP6 (SEQ ID No. 26, 5'-ATTTAGGTGACACTATAG- 3') PCR was carried out. The PCR reaction conditions were the same as those for ITS. After electrophoresis analysis, the optimal concentration of plastids in the hybrid control group (1 ng/μl) was selected (Fig. 3).

Nylon membrane wafer hybridization (Chip hybridization)

Place each nylon membrane wafer finished in a 24-well cell culture dish and add 0.5 x SSC [1 x SSC 0.15 M sodium chloride (FSA Laboratory Supplies, Loughorough, England), 0.015 M sodium citrate, pH 7.0. (Sigma)] -0.1% SDS, washed twice at room temperature (rotational oscillation on a rotary shaker, 75 rpm) for 3 minutes each time to remove trace dye. At the same time, the target DNA of the strain to be identified is amplified by PCR, and 5 μl is added to 200 μl of the hybrid solution together with 2 μl of the hybrid control sample [hybridization solution, 5×SSC, 1% (w/v). Blocking reagent (Roche), 0.1% N-laurylsarcosine (Sigma) and 0.02% SDS] were heated at 95 ° C for 6 minutes to denature the DNA and the double helix was opened. Single strands and immediately placed on an ice bath for 10 minutes to maintain a single strand. The aforementioned wafer was mixed with the denatured target DNA product and the hybrid control sample into a 24-well cell culture dish (Techno Plastic Products, Trasadingen, Switzerland), and the hybrid reaction was carried out at 50 ° C, 120 rpm in a hybrid reaction chamber. hour. The nylon membrane wafer after the completion of the hybridization reaction was washed four times with 0.25 x SSC-0.1% SDS buffer at 60 ° C for 5 minutes to wash away the residual hybrid probe. Then add a blocking buffer (dissolved in maleic acid buffer [0.1M maleic acid (Sigma) and 0.15 M NaCl], 1% (w/v)} diluted 1000 times the egg white A-alkaline phosphatase (strepavidin-AP) was allowed to stand at room temperature for 1 hour. Add 200 μl MAB Wash Buffer [MAB Wash Buffer, 0.3% (v/v) Tween 20, in maleic acid buffer], wash twice at room temperature for 15 minutes, wash away unbound egg white Au-alkaline phosphatase. Add 200 μl of assay buffer (assay buffer, 0.1 M Tris-HCl, 0.15 M NaCl, pH 9.5) for 1 minute at room temperature and remove the assay buffer. NBT/BCIP (nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate, Roche) was used for detection buffer The solution was diluted 50 times and mixed uniformly. The above-mentioned nylon membrane was placed in a 24-well cell culture tray, and 100 μl of the mixture was added dropwise to the mixture at room temperature for 30 minutes in the dark, and the reaction was not shaken. After the color reaction is completed, it is washed four times with sterile water, and the remaining NBT/BCIP on the film is washed. After coloring, the nylon film is placed at room temperature to avoid drying. A high-resolution scanner (Umax powerlook 3000, Taipei, Taiwan) was scanned to store images of the hybrid reaction.

Interpretation of wafer hybridization results

The nylon membrane reaction results can be directly visually interpreted, with coordinate marks on the wafer, dividing the wafer into four regions. The position of the probe in which the reaction takes place is determined by the position at which the hybrid reaction occurs. In the positive control group, a signal is generated, indicating that the target DNA of the detection is successfully increased, and the hybridization control must also generate a signal, indicating that the heterozygous reaction is effective during the heterozygous process, and negative. The control group should have no signal.

Example 2: Lactobacillus ( Antrodia Spp.) wafer performance test Antrodia Analysis of gene sequences within the genus

The length of ITS1-5.8S-ITS2 rDNA product obtained by amplifying 15 kinds of Antrodia spp. , 4 kinds of Antrodiella spp. and 1 kind of Pycnoporus sanguineus by using universal primers ITS4e and ITS5e About 600-700 bps, including a small part of the 18S back, a small sequence of ITS1, 5.8S, ITS2 and 28S front ends. The sequence obtained by sequence analysis of the amplified products was subjected to sequence analysis by Vector NTI 9.0 software to obtain an ITS identity similarity of 55-98%.

Design and test results of specific probes

After ITS sequence and ratio analysis using Vector NTI 9.0, the search position was in the region of high dissimilarity between ITS I and ITS II, and used as the candidate sequence for designing the probe, and simultaneously analyzed the sequence conditions using the oligo analyses function of Vector NTI 9.0. Successfully designed specific probes for 15 kinds of Antrodia spp., 4 kinds of Antrodiella spp. and 1 kind of P. sanguineus . In the probe design process, since A. alpina and A. xantha have very little difference in the ITS sequence (similarity 97%), in order to distinguish the two strains, the sequence of the probe is shortened to distinguish the two. The length of the strain, A. alpina and A. xantha probes are 30 bp, and only 6 bp difference in these probes can distinguish the two species. Antrodiella americana and A. semisupina have very different dispersion regions in the ITS sequence alignment, so the design method is the same as above, and shorter probes can be used to distinguish them. A. salmonea and A.cinnamome have a similarity of only 85% in the ITS sequence, but can be designed on A. salmonea to distinguish the difference regions of the two strains, most of which have multiple repeats of the same base, so after hybridization It is easy to generate non-specific signals, so it is only possible to select the area currently designed. The probe designed in this area is easy to self-bond to form a secondary structure, so the signal is weak after the hybrid reaction, and the above strain is due to the probe. The length is shorter, so the signal after the hybrid reaction is longer and the probe is weaker.

The DNA of all the target strains was amplified by PCR and the target DNA was hybridized with the wafers of all the probe spots. As a result, the probes of the 20 target strains did not produce non-specific hybrids. The combination reaction proves that it can be used to identify the extremely physiologically active and commercial value of Antrodia camphorata, and to identify the various members of the genus Antrodia and the genus Antrodiella caused by brown or white rot. Pathogenic fungi (Figure 4). In addition, the color and morphology of the fruiting body can be distinguished from Pycnoporus sanguineus .

Hybrid reaction of non-target strains

50 common important forest pathogenic fungi and common fungi in soil collected by the strain center and forest test (Table 3), using the same extensive primers to increase the target gene of ITS4e and ITS5e and then hybridize with Antrodia wafer. From the results of the reaction (Fig. 5), it can be seen that only HC and PC appear on the wafer after hybridization with all non-target strains, and the specific probe designed for the Antrodia genus has no signal, and the wafer does. It does not produce non-specific heterozygous reactions with these common non-target strains and is highly specific.

Exploring Needle sensitivity test

The genomic DNA of A. cinnamomea , A. salmonea and P. sanguineus was serially diluted 10-fold at a concentration ranging from 1 ng to 10 fg. Different concentrations of DNA were used as templates to carry out PCR reaction to prepare target DNA and The wafer was subjected to a heterozygous reaction, and the results showed that A. cinnamomea detection limit was 1 pg/μl, A. salmonea detection limit was 10 pg/μl, and P. sanguineus detection limit was 1 pg/μl (Figures 6, 7 and Eight).

Detection of multiple target strain samples

This experiment tests whether the wafer can be used for simultaneous testing of multiple samples of the target strain, two ( A. Americana and A. Cinnamomea ), three ( A. americana , A. Cinnamomea and A. salmonea ) and four ( A. DNA mixing of Americana , A. cinnamomea , A. Salmonea and A. xantha ) fungi, followed by PCR to prepare the target DNA for hybridization with the wafer, the results show that the wafer can be used for multiplex detection applications, but due to the added target strain DNA species The more PCR, the template DNA will compete with each other for the primer pair, and the concentration of the target DNA product of each pathogen will increase, so the more the number of detected bacteria can be found by the signal display, the intensity of the signal will gradually weaken. (Figure 9).

Detection and identification of microchips in forest samples and fruiting bodies

A. cinnamomea from different sources, including commercially available products, samples collected from Xueshankengxi, and P. sanguineus fruiting bodies collected from the experimental forest farm of Yilan University, were extracted by CTAB-small DNA extraction method. DNA, then use PCR to amplify the target DNA. By electrophoresis analysis, a specific product of about 700 bp can be seen, and these products can be heterozygously reacted with the Antrodia wafer to obtain a specific signal generation, and the signal also indicates the position of the desired species to be detected (Fig. ten).

Sequence analysis and alignment analysis of forest tree samples and fruiting body PCR products

In order to determine the correctness of the signal presented after the hybridization, the collected PCR product of the fruiting body sample is subjected to TA selection, and then sent to the biotechnology company for sequencing, and the obtained sequence is uploaded to the National Institutes of Health genetic database ( GenBank, NCBI, http://www.ncbi.nlm.nih.gov/ ). The comparison results show that the ATS sequence of A. cinnamomea is the most similar species in the NCBI gene database. A. camphorata , which is the old name of Antrodia, is actually A. cinnamomea , so the order is compared. The results are in agreement with the wafer test results.

<110> National Taiwan University

<120> An oligonucleotide biochip for identifying Antrodia and Antrodiella and method thereof

<130> 0873-NTU-TW

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<170> PatentIn version 3.5

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<213> Antrodiella liebmannii

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<213> Antrodia albobrunnea

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<213> Antrodia alpina

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<213> Antrodia malicola

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Figure 1 The detailed development process of the biochip of the present invention. (A) is a cartoon icon. (B) Explain the text.

Figure 2 shows a graphical representation of the design of Antrodia and similar nylon membranes and plastic oligonucleotide biochips. The light blue cross is the position marker (PM), the lower right corner is the hybridization control (HC), the positive control group (PC), and the cross center is the negative control group (negative control, NC). The other positions are the positions that are placed by the specific probe.

Figure 3. Hybridization control preparation process. CoKS1 (5'-TTCAACATGTCTCCCCGTGAGG-3', SEQ ID No. 21) and CoKS2 (5'-AAGGGGGTCGACACCAGCATCGTCG-3', SEQ ID No. 22) were used for the DNA of Colletotrichum graminicola using a broad-spectrum primer. PCR amplification was carried out, the amplified product was selected, and the selected plastids were extracted and serially diluted, and PCR was performed on SP6 and T7 by primers. After electrophoresis analysis, the optimal concentration of plastids in the hybrid control group was selected. (1 ng/μl).

Figure 4. Hybrid signal interpretation of the oligonucleotide nylon membrane biochip, which can accurately identify 15 species of Antrodia , 4 Antrodiella , and Pycnoporus. Sanguineus ).

Figure 5. Hybridization of Antrodia oligonucleotide biochips with non-target strains, showing no cross-hybridization of non-specificity.

Figure 6. Sensitivity test of Pycnoporus sanguineus specific probe, the results show that the limit is 1 pg.

Figure 7. The sensitivity test of Antrodia cinnamomea specific probe showed a limit of 1 pg.

The sensitivity test of the specific probe of Antrodia salmonea showed that the limit was 10 pg.

Figure 9 uses a biochip to simultaneously detect multiple Antrodia . (A) Antrodiella americana + Antrodia cinnamomea ; (B) Antrodiella americana + Antrodia cinnamomea + Antrodia salmonea ; (C) Antrodiella americana + Antrodia cinnamomea + Antrodia salmonea + A. xantha .

Figure 10. Application of biochip for diagnosis and identification of Antrodia cinnamomea and Pycnoporus sanguineus . The extracts of A. camphorata and Hemophilus edulis were collected from different regions, and the nucleic acid was extracted. The quality of the purified nucleic acid was confirmed by colloidal electrophoresis, and then amplified by the broad-acting primer pair of the intracellular transcribed region (rDNA) and performed with the biochip. Inverse hybridization, the heterozygous point presented by the color signal after hybridization, can be visually discriminated, and the two species can be clearly identified and distinguished. This identification process can be completed in seven hours.

Claims (6)

A biochip useful for identifying Pycnoporus sanguineus , Antrodia , and Antrodiella , comprising a plurality of probes immobilized on the biochip, wherein the probe comprises An oligonucleotide sequence for identifying M. erythropolis, an oligonucleotide sequence for identifying a genus Phaeoporococcus, and an oligonucleotide sequence for identifying a genus of the genus Phaeobacterium, wherein the probe is The sequence consists of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8. SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, Oligonucleotide sequences of SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20 and their fully complementary sequences. The biochip according to claim 1, wherein the sequence of SEQ ID No. 1 and a probe of the complete complement thereof are used to identify Pycnoporus sanguineus . The biochip according to claim 1, wherein the sequence of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5 and a probe thereof are completely complementary. It is used to identify species like the genus Antrodiella . The biochip according to claim 1, wherein the SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 or SEQ ID The sequence of No. 20 and the probe of its fully complementary sequence are used to identify species of the genus Phaeoporococcus. A method for identifying a genus Hemophilus, a genus of the genus, and a genus of the genus, comprising the steps of: (a) extracting deoxyribonucleic acid (DNA) from a strain to be tested; (b) using a forward primer as a SEQ ID NO: 21 and a reverse primer are the primer pairs of SEQ ID NO: 22 to extract the sequence of ITS1-5.8S-ITS2 ribosomal DNA (rDNA) fragment in the DNA by amplification; (c) the product of step (b) The biochip described in claim 1 is subjected to a hybrid reaction; and (d) determining the genetic species of the test strain by observing which product of the step (b) has a positive reaction with a probe on the wafer. The method of claim 5, wherein the product of step (b) has the SEQ ID No. 1 oligonucleotide sequence or the complementary strand thereof in the biochip of claim 1 In the case of a positive reaction, the strain to be tested is identified as Pycnoporus sanguineus ; the product of the step (b) is the SEQ ID No. 2 oligonucleotide in the biochip described in claim 1 If the sequence or its complementary strand has a positive reaction, the strain to be tested is identified as Antrodiella americana Ryvarden et Gilbertson; the product of step (b) is in the biochip described in claim 1 The SEQ ID No. 3 oligonucleotide sequence or its complementary strand has a positive reaction, and the strain to be tested is identified as Antrodiella formosana Chang et Chou; the product line and patent application of step (b) The SEQ ID No. 4 oligonucleotide sequence in the biochip described in the above paragraph 1 or the complementary strand thereof has a positive reaction, and the strain to be tested is identified as a tough bacterium ( Antrodiella liebmannii (Fries) Ryvarden ); the product of step (b) and the patent application A first oligonucleotide sequence SEQ ID No.5 or the complement of the shares of biochips has a positive reaction, then the test strain is identified like a thin semi Yang volvatus (Antrodiella semisupina (Berkeley et Curtis) Ryvarden); the product of step (b) has a positive reaction with the SEQ ID No. 6 oligonucleotide sequence or its complementary strand in the biochip of claim 1 of the patent application, and the test strain is identified as Antrodia albida (Fries) Donk; the product of step (b) is positive with the SEQ ID No. 7 oligonucleotide sequence or its complementary strand in the biochip described in claim 1 In the reaction, the strain to be tested is identified as Antrodia albobrunnea (Romell) Ryvarden; the product of the step (b) is SEQ ID No. 8 in the biochip described in claim 1 If the oligonucleotide sequence or its complementary strand has a positive reaction, the strain to be tested is identified as Antrodia alpina (Litschauer Gilbertson et Ryvarden); the product of step (b) and the scope of claim 1 SEQ ID No. 9 oligonucleotide sequence in the biochip or its complementary strand If there is a positive reaction, the strain to be tested is identified as Antrodia carbonica (Overholts) Ryvarden et al.; the product of step (b) and the SEQ in the biochip described in claim 1 If the ID No. 10 oligonucleotide sequence or its complementary strand has a positive reaction, the strain to be tested is identified as Antrodia cinnamomea Chang et Chou; the product of step (b) is described in item 1 of the patent application scope. The SEQ ID No. 11 oligonucleotide sequence or its complementary strand in the biochip has a positive reaction, and the strain to be tested is identified as Antrodia juniperina (Murrill) Niemelä et Ryvarden; step (b) The product is identified as a positive reaction with the SEQ ID No. 12 oligonucleotide sequence or its complementary strand in the biochip described in claim 1 of the patent application, and the strain to be tested is identified as a larvae TFRI 550 ( Antrodia lalashana Chang et Chou (TFRI 550)); the product of step (b) has the SEQ ID No. 13 oligonucleotide sequence in the biochip of claim 1 or its complementary strand Positive reaction, the strain to be tested is identified as L. glabrata TFRI 1 607 ( Antrodia lalashana Chang et Chou (TFRI 1607)); the product of step (b) and the SEQ ID No. 14 oligonucleotide sequence or the complementary strand thereof in the biochip of claim 1 has positive In the reaction, the strain to be tested is identified as Antrodia malicola (Berkeley et Curtis Donk); the product of the step (b) is SEQ ID No. in the biochip described in claim 1 of the patent application. If the 15 oligonucleotide sequence or its complementary strand has a positive reaction, the strain to be tested is identified as Antrodia oleracea (Davidson et Lombard) Ryvarden; the product of step (b) and the scope of patent application 1 The SEQ ID No. 16 oligonucleotide sequence or the complementary strand thereof in the biochip has a positive reaction, and the strain to be tested is identified as Antrodia salmonea Chang et Chou; step (b) The product is positively reacted with the SEQ ID No. 17 oligonucleotide sequence in the biochip described in claim 1 or its complementary strand, and the strain to be tested is identified as Antrodia serialis (Fries: Fries) Donk); the product of step (b) and the patent application The biochips in item 1 of SEQ ID No.18 or a nucleotide sequence complementary to an oligonucleotide shares a positive reaction, then the test strain identified as dirty thin volvatus (Antrodia sordida Ryvarden et Gilbertson); step ( The product of b) has a positive reaction with the oligonucleotide sequence of SEQ ID No. 19 in the biochip described in claim 1 or its complementary strand, and the strain to be tested is identified as a bacterium of the genus Taxus ( Antrodia taxa Chang et Chou); or the product of step (b) has a positive reaction with the SEQ ID No. 20 oligonucleotide sequence in the biochip described in claim 1 or its complementary strand, The strain to be tested was identified as Antrodia xantha (Fries) Ryvarden.