TW201020326A - Rapid inspection method on microorganism community structure in anaerobic hydrogen producing system - Google Patents

Abstract

This invention is about method that enables rapid inspection on microorganism community structure in anaerobic hydrogen producing system, with steps described as following: (a) acquiring a sample from anaerobic hydrogen producing system, (b) extracting RNA and DNA from the sample, (c) retrotranscribing RNA with reverse primer of specific primer set to yield cDNA mixture, (d) conducting PCR using the cDNA mixture in (c) and relevant forward primer; meanwhile, perform PCR with DNA in (b) and either one direction of the specific primer set, and (e) observing the amount of PCR product generated in (d) to evaluate the microorganism community structure in anaerobic hydrogen producing system. In addition, this invention also covers the primer set for retreotranscription and PCR, and its usage in DNA or RNA quantification.

Description

201020326 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method for rapidly analyzing the microbial and community structure of an anaerobic hydrogen production system, mainly by using primers to amplify hydrogen production by microorganisms of an anaerobic hydrogen production system. Enzyme genes to quickly analyze the structure of the community. % Ο [Prior Art] The unit weight of hydrogen is 25 times that of methane, 2.75 times that of petrochemical materials, and the combustion of hydrogen produces only water without other air pollutants, such as carbon monoxide and carbon dioxide. Development is not, one of the important petrochemical alternative energy sources. As humanity is facing a crisis of gradual exhaustion of petrochemical resources, the fossil energy reserves are gradually becoming scarce, and Taiwan's research teams are actively developing a fermentation system that uses waste to carry out biological production, and gradually develops various anti-fresh. However, the stability of these systems is related to the changes in the structure of the internal microbial group. The current research on the community structure of hydrogen production systems is based on existing patent instruments or techniques: The DGGE technology, the clone library and the flue in situ technology (FISH) are used to analyze the existence of the (4) first two materials method for monitoring and analysis, and the analysis process is more than necessary. Waiting for 'if the hydrogen production system is unstable,' and quickly understand the cause that the system damage is difficult to save. 201020326 At present, the microbial community structure analysis method for general anaerobic hydrogen production requires amplification of the hydrogen production gene of the microbial community via polymerase chain reaction (PCR), followed by analysis by DGGE, T-RELF, and selection database, and then sequencing. analysis. It takes at least two hours to analyze the above steps, and it takes at least one week to complete the microbial community structure analysis. It is impossible to immediately monitor the microbial community structure in the system, so it is impossible to adjust the correction of the puerperal system. problem. SUMMARY OF THE INVENTION In view of the absence of the prior art, the object of the present invention is to provide a method for rapidly analyzing the microbial community structure of an anaerobic hydrogen production system, mainly by extracting a nucleic acid of a hydrogen-producing microorganism in a sample, and amplifying the gene. Fragments and analysis of their microbial community composition. ...Because of the anaerobic hydrogen production system used in the industry, the variability of the structure of the falling structure causes instability in the operation of the hydrogen production system, so whether the composition of the microbial community can be effectively detected, that is, It becomes an important factor in the treatment of hydrogen production system, which means that rapid detection can shorten the time of crisis treatment and achieve better hydrogen production efficiency. In order to analyze the composition of the microbial community to understand the effects of various microbial changes or attenuation, it can be analyzed by using the differences in specific gene fragments of different microbial communities. To achieve the above object, the present invention provides a primer pair for the production of a hydrogenase gene of a microorganism species, comprising: γ〇: a nucleotide sequence of 1 to 16 and its complementary strand. A method for rapidly detecting microbial population structure 201020326 in an anaerobic hydrogen production system using a pair of primers is also provided. [Embodiment] The method for rapidly detecting microbial community structure in an anaerobic hydrogen production system according to the present invention is characterized by being simple, effective, and rapid, and includes the following steps: (a) providing an anaerobic hydrogen production system (b) separately extracting the RNA and DNA of the aforementioned sample; (c) taking the RNA obtained in the above step (b), and performing reverse transcription with the reverse primer of one of the primer pairs 1 to 2 to obtain a cDNA mixture; Wherein the primer pair 1: SEQ ID NO: 1, SEQ ID NO: 2; primer pair 2: SEQ ID NO: 3, SEQ ID NO: 4; (d) respectively added to the mixture obtained in the aforementioned step (c) The positive primer is subjected to a polymerase chain reaction; the DNA obtained in the above step (b) is simultaneously subjected to a polymerase chain reaction with one of the primer pairs 3 to 8; wherein the primer pair 3: SEQ ID NO 5, SEQ ID NO 6; primer pair 4: SEQ ID NO 7, SEQ ID NO 8; primer pair 5: SEQ ID NO 9, SEQ ID NO 10; 201020326 primer pair 6 · SEQ ID NO: 11, SEQ ID NO: 12; : SEQ ID NO: 13, SEQ ID NO: 14; Of the sub-8: SEQ ID NO.- 15, SEQ ID NO: 16; and (e) the observation step (d) the amount of product to determine the microbial community structure Lu anaerobic hydrogen production systems. In a preferred embodiment, the sample is a biofilm of sludge, compost, sewage or wastewater system; wherein the sludge is preferably a biological sludge. The aforementioned biological sludge refers to a sludge containing microorganisms, and the sludge source is generally produced by a wastewater treatment system. In a preferred embodiment, the polymerase chain reaction of step (d) above is an instant quantitative polymerase chain reaction. In a preferred embodiment, the sample of the anaerobic hydrogen production system is preferably an anaerobic sputum microorganism; a better genus microorganism, • a Bifidobacterium genus, a K!ebsieHa genus micro-branche, and a Pseudomonas genus _ or Streptococcus genus micro-branches; the most funeral, Clostridium pasteurium, Clostridium butyricum gas Klebsiella pneumoniae. To achieve the above object, the present invention provides a primer pair for detecting a microbial community structure in an anaerobic hydrogen production system, which is: primer pair 1: SEQ ID NO: 1, SEQ ID NO: 2; primer pair 2: SEQ ID NO: 3, 201020326 Primer pair 3: SEQ ID NO: 4; SEQ ID NO: 5, primer pair 4: SEQ ID NO: 6; SEQ ID NO: 7, primer pair 5: SEQ ID NO: 8; SEQ ID NO: 9 , primer pair 6 : SEQ ID NO: 10; SEQ ID NO: 11 > primer pair 7 : SEQ ID NO: 12; SEQ ID NO: 13 > SEQ ID NO: 14; or primer pair 8: SEQ ID NO: 15, SEQ ID NO: 16; or its complementary strand. In a preferred embodiment, the sample of the anaerobic hydrogen production system is preferably an anaerobic hydrogen producing microorganism; more preferably a C7〇yir/^wm genus, a Bifidobacterium genus, a Klebsiella genus, ❹ The household (10) is still a microbe or a Sirepiococcw· microorganism; finally you are Clostridium pasteurium, Clostridium butyricum or

Klebsiella pneumoniae. The present invention also provides a use of a scorpion as described above for the quantification of DNA or RNA; more preferably, the aforementioned pair of primers are pairs of primers or primers. The following embodiments are intended to further understand the advantages of the present invention and are not intended to limit the scope of the invention. "201020326 Example 1 Example, extracting the biological nucleic acid in the biological biological sludge in the sputum system by PCR. The anaerobic hydrogen production system used in this embodiment is a self-designed glass device, which is A Continuous Stirred Tank Reactor (4 liters in actual reaction volume). A suitable amount of sludge was collected under standard conditions (1 atm, 25 ° C). Samples were directly taken from the biological sludge grown in the reaction tanks of the following different culture conditions and numbered as 'sample 1-5: sample 1: starch as matrix (carbon source), reaction tank HRT (hydraulic force) The retention time refers to the time during which the water molecules stay in the reaction tank, where the time during which the microorganisms stay in the reaction tank, that is, the incubation time of the biological sludge, is controlled at 12 hours, and the pH is 6.0. . Sample 2: Xylose was used as a substrate, HRT was controlled at 12 hours, Lu pH was 6.0, and powder active carbon (PAC) was added as a cell carrier. The cell support function used herein provides the adhesion and aggregation of microorganisms on the support to increase the concentration of the cells in the reaction tank, thereby improving the effectiveness of the analysis of the bacterial population structure. Sample 3: Glucose was used as a substrate, HRT was controlled at 1 hour, pH was 6.0, and sillicon gel cell (SC) was added as a cell carrier. Sample 4: Xylose was used as a substrate, HRT was controlled at 4 hours, pH was 8 201020326 6.0, and an immobilized cell mass (IM) was added. The immobilized cell carrier used herein is a synthetic silicone gel which utilizes pores in the silicone to trap microorganisms in the pores of the silicone. The matrix, reactants and products in the external environment are freely accessible. The fine pores inside the silicone are free to enter and exit to participate in the reaction. Sample 5: Xylose was used as a substrate, HRT was controlled at 0.5 hour, pH was 6.0, and an immobilized cell carrier was added.调整 Adjust the OD_ absorbance of the sludge to 〇.6_1〇 (if 〇d6〇〇 is outside this range, dilute with sterilized ultrapure water), take 2〇〇μΕ sludge' to Blood & Tissue Genomic DNA Extraction Miniprep System (VIOGENE, Taiwan) extracted its DNA; another 1 mL of sludge was extracted from SV Total RNA Is〇la plus n (10) (卩11〇]^£0 八,1;8 八) .

Primer Design Hydrogen microorganisms The primer pairs used in the present invention are designed for anaerobic production of hydrogenase genes, as shown in the following table j. 201020326 si i spiders δ ^ • S δ ^ 3 ·*»* 〇V ·* ** 2 ^ u S _ V Is •is, T »«* "·» k 3 %%¥ o 廷激 1 ^ JaJL sul. ^5® δ tl ^ s C 〇端' CO u SS | Co 00 匕, 〇q W 蹲® a 4 s: 1^ ^ -cu CO >> v〇...—CO m (Ν q .2 ® 3 §砩c〇δ <; C〇S 〇® S硝 a - c〇S o 2 2 CN Oi 〇τ-Η 1 Ο »r> CO X jg. o 卜 l o v-> inch l jg. 〇m inch loo CN lo (N t**H l O o cn l /*—N rf.tJ Forging: S| S_^ /—\ ό-^6 Q ^ α9 do xn m ^ 1 〇〇cn Η Λ 5[: < ί ^ H ^ ϋ 〇&lt ; go ^ HH cj ^ O ^ H < H < 〇^ OHL·-, £h ^ u 〇H < u 1 1 i〇/—V m 〇Q >~H /—N α inch w 6 h Q 1 1—< 8c/ o^S ϋ u -< E-1 Sy u 5 2u <! ^ 〇S <3 H CH ϋ < HUHU < u < o 1 1 in i〇 /·-N IT) 6 ^ V〇Q .. ^ 〇巴 sk ex Λ ω < CO < w H ?〇< ' U < ϋ ou〇< ^ Η H < 〇 <3 HH 〇H h << O <<c H Sy ο P < u < HCH 1 1 U〇/-s 卜• · /-s 〇00··· \ 1 〇S ^ a〇w WW ΰ ^ o . c 〇〇^ 〇3 o << 〇ϋ o 〇h 〇〇〇g Η ϋ CUH < OC CO Age 1 yr\ m /—\ o ^〇 6 Q ^ c / P w ^ CO cyw whl/l 『 H *7 o C u 〇H Η HC 〇HU Ο H ϋ h ο O ϋ HH 〇η ϋ Ο HUU ϋ < ϋ ο ϋ 〇υ ϋ Η ο Η Η 1 1 ^ CM t—H §6 9 Q hH O* Qi WW ''—^ 〇ΰ E—1 ϋ uh υ og H ^ o 2 u 〇σ <d 2〇2< u ^ 5 << H <:U 1 ! cn ^ —— ..· · 〇〇〇a Η-i ^ cyO" WW «X ^ cn << cu 〇<< H 〇CCU c rS^〇9 〇< u 〇< o 〇<<〇〇< H Η H ^ 〇< tj 〇< CH 0 u 1 1 »ni〇/—N v〇>< /-~N in · ·〇-^ 2臼Q 〇^ QQ <yc/3 W ww^> Today s H <! ο oou CH Η H < ϋ 〇c H <3 ◦ ϋ HO 〇^ o 2 &lt ; <〇< u O u H <ϋ匕〇<3 HU 1 1 U^im Drawing < X) o P. 〇1 KU •fy PQ τ3 W Ρη P. rH 00 <N CO 屮糇nr> CN cn inch Ό 卜 oo 01 201020326 Reverse transcription and PCR analysis using the reverse transcription kit lmPr〇m-nTM Reverse Transcription System (PROMEGA, USA), using one of the reverse primers of the extracted excipient pair 1 to 2 described above, wherein the transfer of the aforementioned RNA secondary structure dissociation is carried out using the cDNA 'reported cDNA 2:

Table 2, RNA secondary structure dissociation formula Formulation Addition amount Extracted RNA 1 μΐ Reverse primer 0.5 μΐ Nuclease-free water 3.5 μΐ Total 15 μΐ The formulation mixture of Table 2 was 7 μL. 〇中拉 Pulled in ice water for 5 minutes' to make RNA secondary knot: Wei furnace boat. In the middle, g喆~Where is left, then the reverse transcription step is entered. Among them, the formula used in the reverse transcription step is shown in Table 3, the reverse transcription formula 3 formula, the above-mentioned RNA secondary sputum mixing, and the ribozyme-free water &

rTM 5x Reaction Buffer

ImProm-II MgCl2 dNTP mixture Recombinant RNasin RNase inhibits the total amount of ImProm-IITM reverse transcriptase

The formulation mixture of Table 3 was incubated for 90 minutes at 45 ° C in 25 ° C. + ° for 5 minutes, then with the TM-clock, and finally the sample center clock = 11 201020326 will be the same as the direct extraction of DNA - PCR step. Φ

In addition, a Master mix (Pr〇mega, USA) kit was used for polymerase chain reaction (PCR). Combining the extracted DNA with one of the primer pairs 3 to 8, respectively, or the forward primer of the DNA and primer pair 1 or 2 which has been subjected to reverse transcription, wherein the amount of DNA is controlled by volume in each concentration. The reaction is less than 100 ng, and the amount of the primer is less than 100 ng per reaction. The PCR reaction conditions were two denaturation reactions at 95 ° C and annealing at 5 passages, at 72 °. The elongation was carried out under the armpit for a total of 35 cycles. As a result, the amplified gene fragment obtained by the above-mentioned polymerase chain reaction was subjected to electrophoresis for 25 minutes under a Li voltage, and the electrophoresis result was stained with ErBr nucleic acid dye, for example, as described in Fig. The results of the first diagram are the microbial community names in samples 1-5 taken from the anaerobic hydrogen production system using the primers of the present invention. The milk is shown in Table 4 below: Heart,,,. The result of the structure. The result is also 12 201020326 Table 4 Sample preparation (carbon source / HRT) Species present 1 Starch / 12 hours (pH 6.0) Clostridium butyricum Bifidobacterium Stupid Pseudomonas Μ Klebsiella pneumoniae Clostridium Genus 2 Xylose / 12 hours (PAC) Clostridium butyricum Clostridium pasteurium Pseudomonas M Klebsiella pneumoniae Streptococcus M Clostridium 3 glucose / 1 hour (SC) (granulated sludge) Clostridium butyricum Clostridium pasteurium Bifidobacterium Explosive Klebsiella pneumoniae Streptococcus Genus Clostridium genus 4 xylose / 4 hours (IM) Clostridium Butyricum Bifidobacterium genus Pseudomonas Mt Klebsiella pneumoniae Clostridium blast ----- 5 xylose / 0.5 small _ (IM) (granulated sludge has been formed) Clostridium butyricum Clostridium pasteurium Bifidobacterium genus Klebsiella pneumoniae Streptococcus M Clostridium belongs to another 'and compares the aforementioned polymerization The results of the enzyme chain reaction were compared with the results of conventional DGGE analysis. The following table 5 formula is configured with a denaturing gradient gel. Each DNA sample amplified by PCR 丨-5 by 13 201020326 is injected into a denaturing gradient gel, and then electrophoresed at 80 volts for 12 hours by a denaturing gradient electrophoresis system (from low The concentration was increased to a high concentration, and then stained with EtBr (Ethidium Bromide) to visualize with UV light and to interpret different bright bands on the film (one bright band represents one species), and the results are shown in Table 6. Table 5, Denaturing Gradient Gel Formulation

Denaturing solution 30% 60% 40% Acrylamide/Bis 3 mL 3 mL 5〇χΤΑΕ buffer 0.4 mL 0.4 mL Formamide 2.4 mL 4.8 mL Urea 2.412 g 5.04 g Water to 20 mL 10 % APS 200 mL 200 mL TEMED 20 \\L 20 pL 14 201020326 Table 6. DGGE analysis results sample number (carbon source / HRT) Species present 1 Starch / 12 hours (pH 6.0) Clostridium butyricum Klebsiella pneumoniae Pseudomonas Explosive unknown species xl 2 wood Sugar / 12 hours (PAC) Clostridium butyricum C. celerecrescens Klebsiella oxytoca Pseudomonas M 3 Glucose eight hours (SC) (granulated sludge has been formed) Clostridium pasteurium Klebsiella pneumoniae Streptococcus M Dialister Bifidobacterium hiring unknown species 4 xylose / 4 hours ( IM) Clostridium butyricum Klebsiella pneumoniae Klebsiella oxytoca Pseudomonas M Dialister J% Bifidobacterium genus 5 xylose / 0.5 hour (IM) (granulated sludge produced) Clostridium butyricum Klebsiella pneumoniae Klebsiella oxytoca Dialister Mi C. ce/erecrescews in Table 6 and [for the production of hydrogen bacteria, but in the hydrogen production system Occurrence probability is not high, Z) zWz \ yier genera and genera, compared with non-hydrogen-generating bacteria, and thus detect whether or not there is no substantial economic benefits. The comparison between the rapid detection method of the present invention and the conventional DGGE analysis method is shown in Table 7 below: 15 201020326 Table 7 Introduction Pairs ^ Number Sample 1 2 3 4 5 6 7+8 1 + — + (+) + + — 2 + (+ ) + — + (+ ) (+ ) 3 (+) + + + — + + 4 + — + + + + — 5 + (+) + (+ ) — + (+)

In Table 7, the wales indicate that the extracted nucleic acids are from different samples, and the rows indicate the number of primer pairs used, where the pair 1 and 2 are directed against Clostridium butyricum anti-Clostridium pasteurium. The bows are paired with C3. It belongs to the first flora or the second flora; the primer pair 4 is directed to the genus microorganism; the primer pair 5 is directed to the genus microorganism; the primer pair 6 is directed against the iadWe/Za primer pair 7 and 8 against the 汾reeococcM·? . In addition, the symbol "+" indicates that the presence of the microorganism in the sample is detected by DGGE and the method of the present invention; the symbol "(+)" indicates that the presence of the microorganism in the sample cannot be detected by DGGE, but the present invention The method can detect the presence of the microorganism in the sample; the symbol "one" indicates that the presence of the microorganism in the sample cannot be detected by the DGGE and the method of the present invention. In other words, the symbol "(+)" indicates that the method of the present invention is more acute than the conventional method. It can be seen from the above that the method of the present invention is more sensitive than the conventional DGGE method; in addition, the analysis time of the method of the present invention is also faster, and it takes about 2-3 working days for the DGGE electrophoresis to be obtained from the sample, and the invention 16 is utilized. The method of 201020326 can be completed within 1 working day (within 6 hours). Example 2 'Analysis of microbial community structure in the hernia pupa system

The extraction method of microbial nucleus in sludge sludge P DN A and RN A is as in example i, but the sample used is in a 300L anaerobic fermentation reaction tank under standard conditions (1 atm, 25. Mud's culture condition is: sucrose as a substrate (carbon source), continuous culture for 60 days (HRT = 12h operation for 30 days, HRT = 8h operation for 10 days, HRT = 4 operation for 15 days and finally re-turn Operation for HRT = 8 h for 5 days.) 1 Transcription and immediate determination. Using the primer pair described in Table 1, the reverse transcription and PCR analysis were carried out by the method disclosed in Example 1, but the PCR analysis was changed. LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche) kit for real-time quantitative polymerase chain reaction (real_time PCR). The above 10 extracted DNA and primer pair 3 to 8 respectively, or reverse transcription as described above The DNA and primer pair 1 or 2 forward primer, wherein the amount of DNA is controlled by volume in a concentration of less than 100 ng per reaction. The amount of primer used is less than 100 ng per reaction. The PCR reaction conditions are at 95 °. Denature reaction at C, at 54 ° C Line bonding (annealing), elongation at 72, a total of 35 cycles. This set of systems in the reaction solution added fluorescent dye SYBR Green I for immediate quantification, and double-stranded DNA A substance that is chimeric and emits fluorescence. Currently, the most commonly used fluorescent dye is 8 丫 - - green I, SYBR-green I will be embedded in the small groove between the double strands of DNA 17 201020326 (minor groove), In addition, the fluorescence is released; therefore, the more double-stranded DNA produced by pCR, the more SYBR_green! is embedded, and the stronger the intensity of the released fluorescence is. The results of the above-mentioned real-time quantitative PCR are shown in the second figure. In the quantitative system, the amplified PCR amplification fragment is further cultured in a coliform by a vector (Vect〇r), and a plastid with a gene fragment is obtained. [8, as a standard for quantitative analysis, and then utilized. instrument

The SpactroPhotometer (nano-drop, ND-l〇〇〇) determines the number of copies of each gene to determine the regression curve for the calibration curve (regression curve) of the UT value of the instantaneous quantitative PCR. Finally, the above samples can be substituted into the regression formula to calculate the corresponding number of gene copies. This method is a method for the absolute quantification of PCR. The axis of the second graph (A) represents the HRT operation of the reaction tank (ie, the hydraulic retention time, the time each unit of influent water stays in the reaction tank), at 12 hours and 8 hours after HRT (from i2h to 8 h) One day), mid-eighth (eighth in the middle of 8 hours of operation), end of 8 hours (from 8 h to 1 day before 4 h), early 4 hours (from 8 h to 4 h after one day), and 4 hours in the middle ( When the operation is stable for 4 hours, take a little at the middle), at the end of 4 hours (from 彳^ to 8h and increase the substrate concentration to one time before the original concentration), and at the beginning of 8 hours (from 4h to 8h after one day), And the end of 8 hours (the day before the end of the experimental operation of the reaction tank) 'where the aforementioned substrate concentration comprises a nutrient source and a carbon source. The Y-axis of the second graph (A) shows that the primers 3, 4, 5, 6, and 7+8 of the present invention are used to quantify the 07 valence/heart genus of each period of the reaction tank operation by real-time quantitative PCR. Microorganism (introduction pair 3), genus microbe (introduction pair 4), microbe (introduction pair 6), Pseudom surface s genus micro±object (bow} subpair 5) or imprinted (10)(10)(10) genus micro± (introduction pair 7+8) The absolute number in the system, and the hydrogen production rate (HPR), ie the total hydrogen production / (per 18 201020326 unit reaction volume per hour). The second figure (B) shows the use of the primer pair 1 and 2 of the present invention. The C. butyricum anti-C. pasteurianum is characterized by reverse transcription and real-time quantitative PCR, respectively. The instant quantitative PCR was used to quantify the hydrogen production enzyme cj)Na, and the activity of hydrogen production enzyme activity was obtained. Its Y-axis represents the number of mRNA copies carried per ng of jJNA and per uL of sample. The method of the invention can detect the microbial community structure within 6 hours after obtaining the sludge sample, and can quickly know the growth and decline of the microbial community, so that the stable and unstable microbial community structure in the hydrogen production system can be monitored immediately. In summary, the present invention utilizes the hydrogen-related functional genes of microorganisms in the anaerobic hydrogen production system as a target, breaks through the limitation of the traditional anaerobic hydrogen production system microbial community composition detection method, and constructs an instant detection and monitoring system for functional genes. In order to effectively grasp the dynamics of microbial composition in the anaerobic hydrogen production system, various hydrogen production systems can shorten the crisis processing time when problems occur to achieve the best production efficiency. The invention not only can quickly determine the change of the microbial community composition structure, but can solve the problem as soon as possible in the early stage of the system to prevent the damage of the hydrogen production system and even affect the downstream system' to save a lot of time, manpower and money. Hydrogen system condition processing and its related production systems have great economic benefits. All of the features disclosed in this siemen's book may be combined with other methods. Each of the features disclosed in the book may be selectively replaced by features of coincidence, equality or similarity. Therefore, all of the features disclosed in this specification are merely one of the equivalent or similar features, except for the particularly distinctive features. While the present invention has been disclosed in its preferred embodiments, such as JL, it is not intended to limit the scope of the invention, and it is to be understood that those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The first figures (A)-(E) are the results of the detection of 1-8 using the primer of the present invention in samples 1-5 of the anaerobic calf system, respectively. M: DNA molecular weight standard; 1-8: primer pair 1-8. The second panel shows the results of the primer pair of the present invention for use in real-time quantitative PCR. The second figure (Α) shows the absolute number of various microorganisms in the system for each period of operation of the reaction tank using the primers of the present invention; and the second figure (Β) shows the use of the primer of the present invention. Analysis of the hydrogen production performance of C. and C. of biological sludge in the reaction tank for 1 and 2. [Main component symbol description] None

20 201020326

SEQUENCE LISTING <110>Fengjia University<120> Method for rapid detection of microbial community structure of anaerobic hydrogen production system <130> 08P0388 <160> 16 <170> Patentln version 3.4 <210> 1 <211&gt ; 24 <212> should <213>manual sequence<220><223>primer<400> 1 agtgttcaaa tgttggtaaa tgtg 24

<210> 2 <211> 23 <212> Wk <213>Artificial sequence<220><223>Introduction<400> 2 ctcttgtgtt gcctcagtat tag 23 <210> 3 <211> 24 <;212> m <213>Artificial sequence<220><223>Introduction<400> 3 aacttgaaga tagcgaagca gacc 24

<210> 4 <211> 19 <212> DNA <213> artificial sequence <220><223> primer <400> 4 ccaccattta cacatcctc 19 <210> 5 <211> 23 <212> DNA <213> artificial sequence

<220><223>Introduction<400> 5 aaaggragat taataccgca taa <210> 6 <211> 20 <212> DNA <213> Artificial sequence 23 201020326 <220><223> 400> 6 ttcttcctaa tctctacgca <220> 7 <211> 18 <212>leg<213>manual sequence<220><223>introduction<400> 7 agtaatgcgt gaccgacc <210> 8 <211> 16 <212> im <213> artificial sequence

<220><223>Introduction<400>8 gaaccgccta cgagcc <210> 9 <211> 19 <212> DNA <213>Artificial Sequence<220><223>Introduction<400> 9 ttcgggcctt gcgctatca <210> 10 <211> 22 <212> DNA <213> artificial sequence <220><223> primer <400> 10 tcgccactgg tgttccttcc ta <210> 11 <211> 20 <212> DNA <213>Artificial sequence<220><223>Introduction<400> 11 aaggctgagg tgtgatgacg <210> 12 <211> 20 <212> DNA <213> Artificial sequence<212>;220><223>Introduction 201020326 <400> 12 ctacacacca gcgtgccttc <210> 13 <211> 24 <212> DNA <213>Artificial sequence <220><223>Introduction<400> 13 caacattacg agcagccata ccaa <210> 14 <211> 24 <212>dirty<213> artificial sequence <220><223>

<400> 14 cttaccttcc agacggaaca ccag <210> 15 <211> 20 <212> DNA <213>Artificial sequence<220><223>Introduction<400> 15 tcgtgaagtg cgtgatacgt <210><211> 22 <212> DNA <213>Artificial sequence<220><223>Introduction<400> 16 catacccagg tgaagttcca ta

Claims (1)

201020326 VII. Patent application scope: 1. A method for rapidly detecting the microbial community structure in an anaerobic hydrogen production system, comprising the following steps: (a) providing a sample obtained from an anaerobic pupa system; (b) separately extracting the foregoing RNA and DNA of the sample; (c) The RNA obtained in the above step (b) is reverse-transcribed with the reverse primer of one of the primer pairs 1 to 2 to obtain a cDNA mixture; wherein the primer pair 1: SEQ ID NO : 1, SEQ ID NO: 2; primer pair 2: SEQ ID NO: 3, SEQ ID NO: 4; (d) Adding the corresponding forward primer to the mixture obtained in the aforementioned step (c) for polymerase linkage The DNA obtained by the above step (b) is simultaneously subjected to a polymerase chain reaction with one of the primer pairs 3 to 8; wherein the primer pair 3: SEQ ID NO: 5, SEQ ID NO: 6 引 primer pair 4: SEQ ID NO: 7, SEQ ID NO: 8; Primer Pair 5: SEQ ID NO: 9, SEQ ID NO: 10 Primer Pair 6: SEQ ID NO: 11 SEQ ID NO: 12 Primer Pair 7: SEQ ID NO: 13 SEQ ID NO: 14 Primer Pair 8: SEQ ID NO: 15 SEQ ID NO: 16 21 201020326 2. 3. 4. 5. 6. 7. ❿ 8. (6) = = = quantity to determine the aforementioned anaerobic hydrogen production d d:: method, the above sample is as patent application '2 Or wastewater system biofilm. Biological sludge. The method of the invention wherein the sludge is the method of item 1 wherein the aforementioned step (d) is an immediate quantitative polymerase chain reaction. The hydrogen system "method of =" wherein the aforementioned anaerobic X king community is an anaerobic hydrogen producing microorganism selected from the group consisting of Um microtubules, Bifidobacterium genus, Klebsiella ΜM a- al. ^ , The sacred object, the pseudomons of the pseudomonas is a St. coc (10) genus. The object aj please patent the method described in item i, in which the aforementioned anaerobic Clostridium butyricum ^ Clostridium pasteurium. The method according to the item 1, wherein the aforementioned fascination is Klebsiella pneumoniae. One of the methods is for detecting a microbial community level pair in an anaerobic pupa system, and the system is: 、, 镩 引引引子 Pair 1: Introduction Pair 2: primer pair 3 primer pair 4: SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO: 2; 3, 4; 5, 6; 7, 22 201020326 Pair 5: SEQ ID NO: 9, SEQ ID NO: 10; primer pair 6: SEQ ID NO. 11, SEQ ID NO: 12; primer pair 7: SEQ ID NO: 13, SEQ ID NO: 14; or primer Pair 8 : SEQ ID NO: 15, SEQ ID NO: 16; or its complementary strand. 9. The primer pair according to Item 8 of the patent application, wherein the aforementioned anaerobic hydrogen-producing microorganism is Clostridium microorganism, Bifidobacterium 赓±, Klebsiella 餍敫±, Pseudomonas genus microtubule gas Streptococcus genus microtubule Things. 10. The pair of primers according to item 8 of the patent application, wherein the aforementioned anaerobic pupa microorganism is C/oWrzWz'w?« a microorganism belonging to the first flora or the second flora, and the primer pair is an primer For 3.引. The primer pair according to claim 8, wherein the aforementioned anaerobic hydrogen producing microorganism is a microorganism, and the primer pair is a primer pair 4. 12. The pair of primers according to claim 8, wherein the aforementioned anaerobic hydrogen-producing microorganism is a foot/e heart-like (10) (8), and the pair of primers is a pair of primers. 13. The primer pair according to claim 8, wherein the anaerobic hydrogen-producing microorganism is hew momw microorganism, and the primer pair is a primer pair 6. 14. The primer pair according to claim 9, wherein the anaerobic wind-producing microorganism is a microorganism belonging to the SYre/piococcwi1 genus, and the primer pair is a primer pair 7 or 8. 15. The pair of primers described in claim 8 wherein the argon-producing anaerobic 23 201020326 is a Clostridium butyricum, and the JL is a pair of primers. 16. The primer pair according to item 8 of the patent application, wherein the aforementioned anaerobic hydrogen producing microorganism is, and the primer pair is a primer pair 2. 17. Use of a primer as described in claim 8 of the patent application for the quantification of DNA or RNA. 18. The use of claim 17, wherein the aforementioned pair of primers is a pair of primers or a pair of primers. twenty four