PCR primer pairs and probes for adulteration identification of Saussurea involucrata and their application

Abstract

The present invention belongs to the technical field of drug detection, and specifically relates to a PCR primer pair, a probe, and applications thereof for identifying adulteration of Saussurea involucrata. The nucleotide sequences of the PCR primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:3. Based on the PCR primer pair and / or probe for identifying adulteration of Saussurea involucrata provided by the present invention, adulterated Saussurea jellyfish and / or Saussurea spp. in Saussurea jellyfish can be identified accurately and quickly.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a PCR primer pair, probe, and their application for the identification of adulteration in Tian Shan snow lotus. Background Technology

[0002] Tian Shan snow lotus (Saussurea involucrata (Kar. et Kir.) Sch. Bip.) is the dried aerial part of the plant *Saussurea involucrata*, belonging to the genus *Saussurea* in the family Asteraceae. It possesses properties such as promoting blood circulation, tonifying the kidneys, strengthening muscles and bones, and dispelling wind and dampness. Tian Shan snow lotus grows in extremely harsh environments and is a unique and precious medicinal material endemic to the high-altitude regions of western my country. Due to its slow production rate and low natural reproductive capacity, coupled with long-term harvesting, its wild resources are on the verge of extinction, and it is currently listed as a national second-class protected plant. In addition to Tian Shan snow lotus [S. involucrata (Kar. et Kir.) Sch. Bip.], the genus *Saussurea* (Saussurea DC.) includes nearly 400 other varieties, such as *S. laniceps* Hand.-Mazz. and *S. medusa* Maxim., most of which are distributed in the Qinghai-Tibet Plateau region of my country.

[0003] Tian Shan snow lotus is commonly known as "snow lotus," and in traditional Tibetan medicine, *Saussurea medusa* and *Saussurea costatum* are also called medicinal "snow lotus." Due to the scarcity of wild Tian Shan snow lotus resources and the abundance of related species, coupled with the similarity in local names, *Saussurea costatum*, *Saussurea medusa*, and other related species are sometimes confused with Tian Shan snow lotus in the market. Current quality standards for Tian Shan snow lotus are simplistic and lack specificity for species identification within the *Saussurea* genus. Therefore, it is essential to establish a highly efficient, accurate, and specific method for identifying common and easily confused adulterants of Tian Shan snow lotus.

[0004] Quantitative real-time PCR (qPCR) is currently the most commonly used method for quantitative analysis of gene expression levels, and it has been gradually applied in the testing of traditional Chinese medicine in recent years. Locked nucleic acid (LNA)-TaqMan probes are based on TaqMan probes, with some bases modified by LNA. Introducing LNA modification increases the Tm value of the system, greatly improving the probe's specificity and detection sensitivity. Currently, there are no studies on the application of LNA-TaqMan qPCR for the identification of snow lotus. Summary of the Invention

[0005] This invention develops a PCR primer pair and probe for identifying adulteration in Tian Shan snow lotus, which can be used to identify Tian Shan snow lotus.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] The present invention provides a PCR primer pair for identifying adulteration of Tian Shan snow lotus, the nucleotide sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0008] Another aspect of the present invention provides a PCR primer pair and a probe for identifying adulteration in Tian Shan snow lotus. The nucleotide sequences of the PCR primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:3.

[0009] Another aspect of the present invention provides a detection reagent for identifying adulteration of Tian Shan snow lotus, including the PCR primer pair for identifying adulteration of Tian Shan snow lotus as described in the present invention or the PCR primer pair and probe for identifying adulteration of Tian Shan snow lotus as described in the present invention.

[0010] In another aspect, the present invention provides a detection kit for identifying adulteration of Tian Shan snow lotus, which includes the detection kit for identifying adulteration of Tian Shan snow lotus of the present invention.

[0011] Another aspect of the present invention provides a PCR primer pair for identifying adulteration in Tian Shan snow lotus, or the PCR primer pair and probe for identifying adulteration in Tian Shan snow lotus, and their application in the preparation of detection products for identifying adulteration in Tian Shan snow lotus.

[0012] Another aspect of the present invention provides a method for identifying adulterated Saussurea medusa and / or Saussurea pubescens in Saussurea indicum, comprising: (1) extracting DNA from the sample to be tested and the standard sample to obtain DNA templates for the sample to be tested and the standard sample; (2) using the PCR primer pair and probe of the present invention for identifying adulteration in Saussurea indicum to be tested to perform PCR amplification on the DNA templates for the sample to be tested and the standard sample to obtain a first CT value and a second CT value; (3) calculating the difference ΔCT between the first CT value and the second CT value, and identifying the content of adulterated Saussurea medusa and / or Saussurea pubescens in Saussurea indicum based on the absolute value of ΔCT.

[0013] Preferably, in the above identification method, when the absolute value of △CT is ≤4, the content of false Saussurea medusa and / or Saussurea pubescens in Saussurea in Tianshan snow lotus is ≥10%.

[0014] Preferably, in the above identification method, the PCR amplification program includes: 94℃ / 3min, 94℃ / 5sec, 62.5℃ / 30sec, where 94℃ / 5sec and 62.5℃ / 30sec are used.

[0015] The beneficial effects of this invention include at least the following: based on the PCR primer pairs and / or probes provided by this invention for the identification of adulteration in Saussurea involucrata, adulteration with Saussurea medusa and / or Saussurea pubescens in Saussurea involucrata can be identified accurately and quickly. Attached Figure Description

[0016] Figure 1 Amplification curves for Saussurea medusa, Saussurea volumicola, and Saussurea tianshanensis;

[0017] Figure 2 This is the amplification curve used to evaluate the sensitivity of the real-time PCR method of this invention; the red curve represents the DNA concentration at 0.1 ng / μL. –1 sample;

[0018] Figure 3 The image shows the specific amplification curve for detecting Saussurea medusa using the quantitative real-time PCR method of this invention, where the blue curve represents the blank control.

[0019] Figure 4 The specific amplification curves for detecting the woolly snow rabbit using the real-time PCR method of this invention are shown, wherein the blue curve represents the blank control;

[0020] Figure 5 The image shows the specific amplification curve for detecting Tian Shan snow lotus using the quantitative real-time PCR method of this invention, where the blue curve represents the blank control. Detailed Implementation

[0021] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0023] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0024] This invention provides a PCR primer pair for identifying adulteration in Tian Shan snow lotus, the nucleotide sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0025] It should be noted that the PCR primer pair used in this invention can achieve adulteration identification of Tian Shan snow lotus with good specificity and high accuracy. Specifically, the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 are as follows:

[0026] TSXL4138-F(SEQ ID NO:1):5'-CGACACTCTTAATTGATACG-3';

[0027] TSXL4138-R (SEQ ID NO: 2): 5'-CGGTAAAGTTTGGAAGAC-3'.

[0028] This invention also provides a PCR primer pair and probe for identifying adulteration in Tian Shan snow lotus. The nucleotide sequences of the PCR primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:3.

[0029] It should be noted that this invention can also perform fluorescent PCR identification based on PCR primer pairs and probes, enabling more accurate identification of adulteration in Tian Shan snow lotus. Specifically, the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 are as follows:

[0030] TSXL4138-F(SEQ ID NO:1):5'-CGACACTCTTAATTGATACG-3';

[0031] TSXL4138-R(SEQ ID NO:1):5'-CGGTAAAGTTTGGAAGAC-3';

[0032] M4138-P2(SEQ ID NO:3):5'-FAM-tttCcaTtcAttCcctttc-BHQ1-3' (lowercase letters indicate LNA-modified bases).

[0033] This invention also provides a detection reagent for identifying adulteration in Tian Shan snow lotus, including the PCR primer pair for identifying adulteration in Tian Shan snow lotus as described in this invention, or the PCR primer pair and probe for identifying adulteration in Tian Shan snow lotus as described in this invention.

[0034] It should be noted that the PCR primer pairs or PCR primer pairs and probes in this invention can be mixed with detection auxiliary reagents to prepare detection reagents, including buffer solutions, etc.

[0035] This invention also provides a detection kit for identifying adulteration of Tian Shan snow lotus, which includes the detection kit for identifying adulteration of Tian Shan snow lotus described in this invention.

[0036] It should be noted that the detection reagent for identifying adulteration of Tian Shan snow lotus in this invention can be prepared into a detection kit, which is more convenient for transportation and use.

[0037] This invention also provides an application of the PCR primer pair for identifying adulteration in *Saussurea involucrata*, or the PCR primer pair and probe for identifying adulteration in *Saussurea involucrata*, in the preparation of a detection product for identifying adulteration in *Saussurea involucrata*.

[0038] This invention also provides a method for identifying adulterated Saussurea medusa and / or Saussurea fusiforme in Saussurea involucrata, comprising: (1) extracting DNA from the sample to be tested and the standard sample to obtain DNA templates for the sample to be tested and the standard sample; (2) using the PCR primer pair and probe for adulteration identification of Saussurea involucrata in this invention to perform PCR amplification on the DNA templates for the sample to be tested and the standard sample to obtain a first CT value and a second CT value; (3) calculating the difference ΔCT between the first CT value and the second CT value, and identifying the content of adulterated Saussurea medusa and / or Saussurea fusiforme in Saussurea involucrata based on the absolute value of ΔCT.

[0039] In some specific examples, in the above identification method, when the absolute value of △CT is ≤4, the content of false Saussurea medusa and / or Saussurea pubescens in Saussurea in Tian Shan is ≥10%.

[0040] In some specific examples, the PCR amplification program in the above identification method includes: 94℃ / 3min, 94℃ / 5sec, 62.5℃ / 30sec, where 94℃ / 5sec and 62.5℃ / 30sec are the two intervals.

[0041] I. Determination of Primer Pairs and Probes

[0042] The chloroplast genome sequences of *Saussurea involucrata*, *Saussurea medusa*, and *Saussurea laniceps* were downloaded from the National Center for Biotechnology Information (NCBI: https: / / www.ncbi.nlm.nih.gov / ). Through sequence alignment analysis of these three species, using the *Saussurea involucrata* (MH926106) sequence as a reference, 141 single nucleotide polymorphism (SNP) sites shared by the easily confused species *Saussurea medusa*, *Saussurea laniceps*, and *Saussurea laniceps* were screened. Using Beacon Designer 8 software and a proprietary algorithm, the optimal primer pairs for detection were designed. The cytosine (C)>adenine (A) mutation at position 4138 was ultimately determined as the SNP site, and specific probes and primer pairs for *Saussurea medusa* and *Saussurea laniceps* were obtained. The probe (M4138-P2) was purified by high-performance liquid chromatography (HPLC). The specific probe was labeled with a fluorescein group FAM at the 5′ end and a non-fluorescein quencher group BHQ1 at the 3′ end. The probe and primer sequences are shown below:

[0043] TSXL4138-F:5'-CGACACTCTTAATTGATACG-3';

[0044] TSXL4138-R:5'-CGGTAAAGTTTGGAAGAC-3';

[0045] M4138-P2:5'-FAM-tttCcaTtcAttCcctttc-BHQ1-3' (lowercase letters indicate LNA-modified bases).

[0046] II. PCR Amplification Detection

[0047] (I) Extraction of genomic DNA from samples of Saussurea medusa, Saussurea fusiforme, and Saussurea involucrata.

[0048] Samples of Saussurea medusa, Saussurea voilea, and Saussurea involucrata (see Table 1 for details) with accurate origins were collected. 0.1 g of each sample was washed sequentially with 1 ml of 75% ethanol and 1 ml of sterile ultrapure water. The surface moisture was then absorbed, and the samples were ground into fine powder using a ball mill (or liquid nitrogen grinding). 20 mg of each sample was placed in a 1.5 ml centrifuge tube, and nucleic acid extraction was performed using the Plant Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd. to obtain DNA templates.

[0049] Table 1 Sample Information Table

[0050] Serial Number Plant origin Latin name serial number 1 Tian Shan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-01 2 Tian Shan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-02 3 Tian Shan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-03 4 Tian Shan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-04 5 Tian Shan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-05 6 Tian Shan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-06 7 Jellyfish Snow Rabbit S.medusa Maxim. SMXTZ-01 8 Jellyfish Snow Rabbit S.medusa Maxim. SMXTZ-02 9 Jellyfish Snow Rabbit S.medusa Maxim. SMXTZ-03 10 Jellyfish Snow Rabbit S.medusa Maxim. SMXTZ-04 11 Jellyfish Snow Rabbit S.medusa Maxim. SMXTZ-05 12 Jellyfish Snow Rabbit S.medusa Maxim. SMXTZ-06 13 Jellyfish Snow Rabbit S.medusa Maxim. SMXTZ-07 14 Cotton-headed Snow Rabbit S.laniceps Hand.-Mazz. MTXTZ-01 15 Cotton-headed Snow Rabbit S.laniceps Hand.-Mazz. MTXTZ-02 16 Cotton-headed Snow Rabbit S.laniceps Hand.-Mazz. MTXTZ-03 17 Cotton-headed Snow Rabbit S.laniceps Hand.-Mazz. MTXTZ-04 18 Cotton-headed Snow Rabbit S.laniceps Hand.-Mazz. MTXTZ-05 19 Cotton-headed Snow Rabbit S.laniceps Hand.-Mazz. MTXTZ-06 20 Cotton-headed Snow Rabbit S.laniceps Hand.-Mazz. MTXTZ-07

[0051] (II) Fluorescent PCR Amplification Reaction

[0052] The DNA template obtained above was added to the PCR reaction system for fluorescent PCR amplification and CT value analysis. The PCR reaction system is shown in Table 2 below.

[0053] Table 2 PCR reaction system

[0054]

[0055]

[0056] In addition, the PCR amplification reaction program is as follows: 94℃ / 3min, 94℃ / 5sec, 62.5℃ / 30sec, with the 94℃ / 5sec and 62.5℃ / 30sec cycles performed for 40 cycles.

[0057] (III) CT Value Analysis

[0058] PCR amplification products were analyzed using a Bio-Rad CFX96 analyzer. Amplification curves for different samples are shown below. Figure 1 As shown, the results indicate that the probe primers of this invention tested different batches of Saussurea medusa and Saussurea velutipes, and all samples yielded positive results, while different batches of Saussurea involucrata yielded negative results. This further confirms that the primers and probes of this invention can be used to identify adulterated Saussurea medusa / Saussurea velutipes in Saussurea involucrata.

[0059] III. Identification of Adulteration with Saussurea medusa / Saussurea in Tian Shan Snow Lotus

[0060] (I) DNA Extraction

[0061] Take the fine powder from samples of Saussurea medusa, Saussurea voilea, and Saussurea involucrata, weigh them accurately according to the proportions in Table 3, and mix them evenly (total 0.1g); take 20mg, place it in a 1.5ml centrifuge tube, and extract nucleic acid using the Plant Genomic DNA Extraction Kit of Tiangen Biotech (Beijing) Co., Ltd., to obtain DNA template.

[0062] Table 3. Mixing ratio of Saussurea medusa, Saussurea involucrata and Saussurea involucrata.

[0063] serial number Mass of Saussurea medusa / Saussurea involucrata (g) Mass (g) of Tian Shan Snow Lotus adulteration ratio 1 0.001 0.099 1％ 2 0.005 0.195 2.5％ 3 0.005 0.095 5％ 4 0.010 0.090 10％ 5 0.025 0.075 25％ 6 0.050 0.050 50％ 7 0.100 / 100％

[0064] (II) Fluorescent PCR Amplification Reaction

[0065] The DNA template obtained above was added to the PCR reaction system for fluorescent PCR amplification and CT value analysis. The PCR reaction system is shown in Table 4 below.

[0066] Table 4 PCR reaction system

[0067]

[0068] In addition, the PCR amplification reaction program is as follows: 94℃ / 3min, 94℃ / 5sec, 62.5℃ / 30sec, where 94℃ / 5sec and 62.5℃ / 30sec are followed by PCR amplification reaction.

[0069] (III) CT Value Analysis

[0070] PCR amplification products were analyzed using a Bio-Rad CFX96 analyzer. CT values ​​of samples with different adulteration ratios were analyzed, with 100% adulteration of Saussurea medusa / Saussurea involucrata as a positive control. The relationship between the absolute value of the difference in CT values ​​between samples with different adulteration ratios and positive samples (ΔCT absolute value) and the adulteration ratio was analyzed.

[0071] Given that polymerase chain reaction (PCR) exhibits exponential amplification, the number of amplification rounds required to reach the target value is denoted as the CT value. The difference between the CT values ​​of the standard (S) and the test sample (T) is represented by ΔCT. Therefore, the proportion of the target DNA in the test sample can be expressed as:

[0072] Test sample (T) / Standard sample (S) % = 2 -△CT绝对值 ×100%

[0073] Based on the above formula, the proportion of mass DNA in Saussurea medusa / Saussurea voilea was calculated under different adulteration ratios. The calculation results are shown in Tables 5 and 6.

[0074] Table 5. Relationship between the actual adulteration ratio of Saussurea medusa and the absolute value of ΔCT.

[0075] Mixed samples Ct mean △Ct Adulteration ratio (calculated value) % Adulteration ratio (expected value) % 1 28.40 7.09 0.74 1.0 2 26.83 5.51 2.19 2.5 3 26.16 4.85 3.47 5 4 25.14 3.83 7.05 10 5 23.52 2.21 21.66 25 6 22.22 0.90 53.46 50 7 21.31 0.00 100.00 100

[0076] Table 6. Relationship between the actual adulteration ratio of Snow Rabbit and the absolute value of ΔCT.

[0077] Mixed samples Ct mean △Ct Adulteration ratio (calculated value) % Adulteration ratio (expected value) % 1 28.52 6.84 0.88 1.0 2 27.34 5.66 1.98 2.5 3 26.46 4.78 3.63 5 4 25.21 3.53 8.66 10 5 24.09 2.41 18.77 25 6 22.91 1.23 42.63 50 7 21.68 0.00 100.00 100

[0078] In actual testing, given that medicinal materials are allowed to contain no more than 3% impurities, and taking into full account the possibility of errors in testing, when the actual adulteration is 5%, the ΔCT of Saussurea medusa is 4.85 and that of Saussurea costatum is 4.78. When the actual adulteration is 10%, the ΔCT of Saussurea medusa is 3.83 and that of Saussurea costatum is 3.53. Therefore, an absolute value of ΔCT ≤ 4 is used as the criterion for detection.

[0079] IV. PCR Amplification Accuracy and Sensitivity Testing

[0080] (I) Method sensitivity evaluation

[0081] Saussurea medusa was selected for investigation. A batch of Saussurea medusa genomic DNA was collected and serially diluted with sterile double-distilled water to a DNA template concentration of 100 ng / μL. –1 50 ng·μL –1 20 ng·μL –1 10 ng·μL –1 1 ng·μL –1 and 0.1 ng·μL –1 1 μL of each of the above-mentioned template concentrations was amplified using the PCR conditions constructed above, and the results are as follows. Figure 2 As shown, the results indicate that when the Saussurea medusa template DNA concentration is diluted to 0.1 ng / μL... –1 Even at times, it can still be detected when the Ct value of the amplification curve is ≤32; when the template DNA concentration is below 0.1 ng·μL –1 At that time, the Ct value of the amplification curve was greater than 32. This indicates that the method of the present invention has good sensitivity.

[0082] (II) Method Specificity Investigation

[0083] Using the fluorescent quantitative PCR primers and specific probes of this invention, amplification reactions were performed on 20 batches of *Saussurea involucrata*, *Saussurea medusa*, and *Saussurea laniceps* listed in Table 1 according to the above method to verify specificity. Sterile double-distilled water was used as a blank control. The results showed that the DNA of the adulterants *Saussurea medusa* and *Saussurea laniceps* exhibited typical S-shaped amplification curves, while genuine *Saussurea involucrata* and the blank control showed no amplification, indicating that the primers and probes screened in the experiment have strong specificity. (See...) Figures 3 to 5 .

[0084] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. PCR primer pairs and probes for identifying *Saussurea medusa* and / or *Saussurea costatum* adulterated with *Saussurea involucrata*, characterized in that, The nucleotide sequences of the PCR primer pairs are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:

3.

2. A detection reagent for identifying adulterated Saussurea medusa and / or Saussurea laniceps in Saussurea indicum, characterized in that, Includes the PCR primer pair and probe for identifying adulteration of Tian Shan snow lotus as described in claim 1.

3. A detection kit for identifying adulterated Saussurea medusa and / or Saussurea laniceps in Saussurea indicum, characterized in that, Includes the detection reagent for identifying adulteration of Tian Shan snow lotus as described in claim 2.

4. The application of the PCR primer pairs and probes described in claim 1 for identifying adulterated Saussurea medusa and / or Saussurea fusiforme in Saussurea indicum in the preparation of detection products for identifying adulteration in Saussurea indicum.

5. A method for identifying adulteration of *Saussurea medusa* and / or *Saussurea laniceps* in *Saussurea involucrata*, characterized in that, include: (1) Extract DNA from the sample to be tested and the standard sample to obtain DNA templates for the sample to be tested and the standard sample, respectively; (2) Using the PCR primer pair and probe described in claim 1 for the identification of adulteration of Tianshan snow lotus, PCR amplification is performed on the DNA template of the sample to be tested and the DNA template of the standard sample to obtain the first CT value and the second CT value; (3) Calculate the difference ΔCT between the first CT value and the second CT value, and identify the content of adulterated Saussurea medusa and / or Saussurea fusiforme in Tianshan snow lotus based on the absolute value of ΔCT.

6. The identification method according to claim 5, characterized in that, When the absolute value of △CT is ≤4, the content of false Saussurea medusa and / or Saussurea pubescens in Saussurea in Tianshan snow lotus is ≥10%.

7. The identification method according to claim 5 or 6, characterized in that, The PCR amplification program included: 94℃ / 3min, 94℃ / 5sec, 62.5℃ / 30sec, with a total of 40 cycles of 94℃ / 5sec and 62.5℃ / 30sec.

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