High-conservative primer group for bacterial detection and application of high-conservative primer group

Abstract

The invention discloses a group of highly conservative primers for bacterial detection and application thereof. According to the invention, a group of high-conservative primers for a rapid and accurate exogenous microorganism detection method are developed after efficient comparison and screening are carried out on a microorganism 16S rRNA gene sequence. The method is mainly applied to quality control of cell and gene therapy products. According to the invention, a highly conservative 16S rRNA primer design method is used, and a degenerate base and probability analysis are combined, so that the wide coverage rate and high specificity of detection are ensured; more than 98% of target microorganisms can be covered. The primer designed by the invention has high efficiency and flexibility, can be compatible with a TaqMan probe and an SYBR Green fluorescent dye system according to specific detection requirements, is suitable for various microbiological detection scenes, and ensures the diversity and applicability of detection.

Description

Technical Field

[0001] The invention relates to a group of highly conservative primers for bacteria detection and applications thereof, belonging to the technical field of microbial detection. Background Art

[0002] In the development and production of cell and gene therapy products, exogenous microbial contamination is a key quality control issue. These therapeutic products are directly applied to the human body, and any microbial contamination may cause serious health risks and even lead to adverse consequences such as immune response and infection in patients. Therefore, ensuring the sterility of these products and microbial control during their production has become an important part of drug production and quality control. With the rapid development of the global pharmaceutical industry, emerging therapies such as cell and gene therapy have gradually become the focus of medical research and clinical applications. Especially in cell therapy, due to the operation and application of living cells, the sterility requirements in the production process are extremely strict. Although traditional microbial detection methods have been widely used in the past few decades, they have long detection cycles and low sensitivity, and can no longer meet the needs of rapid detection. Therefore, the development of efficient, rapid and sensitive microbial detection methods has become an urgent need in the industry.

[0003] Internationally, rapid microbiological detection technology (RMM, Rapid Microbiological Methods) gradually emerged at the end of the 20th century. In the industry guidance issued by the US FDA in 2004, it first recommended the use of genetic methods for microbial detection, which are more accurate than traditional biochemical and phenotypic techniques. Since then, countries such as Japan and Australia have also recognized and supported the implementation and validation of rapid detection technologies. The development of these technologies has improved the efficiency of microbial detection, especially in the production of drugs and medical devices. In my country, the 2010 edition of the "Chinese Pharmacopoeia" included the "9201 Guidelines for Validation of Alternative Methods for Microbiological Testing of Pharmaceuticals" for the first time, and proposed the validation and application guidelines for rapid microbial detection methods, but my country still has deficiencies in independently developing rapid microbial detection technologies with independent intellectual property rights. Although the traditional 16S rRNA detection method has a good application in microbial diversity analysis, due to the wide variety of microorganisms, it is always a challenge to design PCR primers with high coverage and specificity.

[0004] Existing primer design methods usually rely on the characteristics of specific bacterial species or groups and cannot meet the detection needs of multiple microorganisms coexisting in complex environments. How to design primers that are both universal and adaptable to the needs of different detection technologies remains a difficulty in research.

[0005] Therefore, there is an urgent need to develop a set of highly conservative primer sequences for rapid detection technology designed using modern bioinformatics, combined with degenerate bases and base probability analysis, to design primers with high coverage and specificity, and with the characteristics of being fast and accurate. They should also be able to adapt to different PCR detection technologies and meet the needs of rapid detection of exogenous microbial contamination in the production process of cell and gene therapy products. Summary of the invention

[0006] The purpose of the present invention is: in view of the shortcomings of the prior art, the present invention provides a group of highly conservative primers for bacterial detection. The present invention comprehensively analyzes the bacterial 16S rRNA gene sequence, screens out highly conservative primers, introduces degenerate bases, and designs a group of highly conservative primer sequences covering more than 98% of bacterial species, which can meet the rapid detection needs of exogenous microbial contamination in the production process of cell and gene therapy products, and the primers can support a variety of detection technologies to ensure the flexibility and accuracy of detection.

[0007] In order to achieve the above object, the present invention provides a group of primers selected from the following group:

[0008] i) any one or more of the primers whose sequences are shown in SEQ ID NOs: 1 to 22;

[0009] ii) any one or more of the full-length primers or primer fragments thereof whose sequences are shown in SEQ ID NOs: 23 to 44. The primers in i) are obtained by screening based on the 16S rRNA sequences of all bacterial species; the primers in ii) are primer sequences after the introduction of degenerate bases.

[0010] The present invention also provides a method for designing the above-mentioned primers, comprising the following steps:

[0011] Step 1: Download the full-length or nearly full-length gene sequences of 16S rRNA of all bacterial species (Beacteria) from the public database Silva database, and perform quality control on the downloaded sequence data to ensure the reliability and accuracy of the data;

[0012] Step 2: Perform consensus sequence analysis on the sequence data obtained in step 1, set filtering conditions (the ratio of "." and "-" is greater than 0.5 and the base ratio is less than 0.01), and draw a base distribution heat map and a similarity line graph to analyze the highly conserved regions of the sequence;

[0013] Step 3: Divide the sequence into continuous sequence segments with a step length of 12-mers in units of 12 bases, and select highly conserved segments according to the following screening criteria to form a candidate sequence set, then connect all continuous 12-mers segments to form a candidate sequence, as shown in SEQ ID NO: 1 to 22;

[0014] The screening criteria are: in each 12-mers fragment, at least 7 bases have a single base probability greater than 0.99, the median probability of 12 bases is greater than 0.991, and 75% of the sites have a base probability greater than 0.995.

[0015] Preferably, the candidate sequence in step 3 is optimized after introducing degenerate bases to obtain primers with sequences as shown in SEQ ID NOs: 23 to 44.

[0016] The present invention also provides the use of the above primers in preparing a reagent or a kit for bacteria detection, wherein the reagent or the kit comprises a primer and / or a probe.

[0017] The present invention also provides a primer, a probe or a primer-probe group for bacteria detection, which is selected from any one or more of the full-length primers or primer fragments thereof whose sequences are shown in SEQ ID NOs: 23 to 44.

[0018] Preferably, the sequences of the primers are shown in SEQ ID NOs: 45-46.

[0019] Preferably, the sequence of the primer probe set is as shown in SEQ ID NOs: 47 to 49, wherein the sequence of the primers is as shown in SEQ ID NOs: 47 to 48, the sequence of the probe is as shown in SEQ ID NO: 49, and the 5' end of the probe is modified with a fluorescent group and the 3' end is modified with a quenching group.

[0020] The present invention also provides the use of the above primers, probes or primer-probe groups in preparing reagents or kits for bacteria detection.

[0021] The present invention also provides a reagent or kit for bacterial detection, wherein the reagent or kit comprises a material selected from the following group:

[0022] i) primers having sequences as shown in SEQ ID NOs: 45-46 and SYBR Green fluorescent dye;

[0023] ii) a primer-probe set having sequences as shown in SEQ ID NOs: 47 to 49, wherein the sequences of the primers are as shown in SEQ ID NOs: 47 to 48, and the sequence of the probe is as shown in SEQ ID NO: 49.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention ensures wide coverage and high specificity of detection by using a highly conservative 16S rRNA primer design method combined with degenerate bases and probability analysis; the present invention compares and analyzes the 16S rRNA gene to find highly conserved and most frequent fragments in bacterial species, ensuring that the designed primers have wide applicability and can cover more than 98% of the target microorganisms; in addition, degenerate bases are introduced in combination with the base distribution of different bacterial species to maximize the application range of the primers, and the degenerate bases greatly improve the coverage and flexibility of detection.

[0026] (2) The primers designed in the present invention are both efficient and flexible. They are compatible with TaqMan probes and SYBR Green fluorescent dye systems according to specific detection requirements and are suitable for a variety of microbial detection scenarios, thus ensuring the diversity and applicability of detection.

[0027] (3) The primer design method of the present invention has good scalability and can be applied to the development of primers for other conserved genes, such as 18S rRNA and ITS regions, by adjusting the screening criteria and parameter settings, thereby meeting the needs of different research fields.

[0028] (4) The present invention provides an efficient and accurate highly conservative primer for bacterial detection, which is suitable for quality control of cell and gene therapy products, environmental microbial diversity analysis, pathogenic microorganism detection, and food and water quality monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1a :The heat map of the full-length four-base distribution of bacterial 16S rRNA gene (total length 1477); This heat map is mainly used to observe the full-length distribution of 16S rRNA genes of all bacterial species. The horizontal axis represents the position of the gene sequence, and the vertical axis represents the base type, including A, T, C, G, and the missing symbols "." and "-".

[0030] Figure 1b :Full-length similarity distribution map of bacterial 16SrRNA gene (total length 1477); By setting the threshold line of ordinate 0.9, the highly conserved and highly variable regions of the sequence can be quickly identified, providing an important reference for primer design.

[0031] Figure 2a : Heat map of bacterial 16S rRNA gene sequences corresponding to the candidate primer sequence positions; the positions of the candidate primer sequences are highly conserved and low in variability in the bacterial 16S rRNA gene.

[0032] Figure 2b: The similarity line graph of bacterial 16S rRNA sequences corresponding to the candidate primer sequence positions. The similarity line graph intuitively shows the conservation of the 22 candidate primer sequences in the bacterial 16S rRNA gene.

[0033] Figure 3a : The number of fragments required for each 12-mers (a fragment with 12 bases from the starting site as a unit) to cover more than 0.99 bacterial species after 12-mers segmentation; There are significant differences in the conservation and coverage efficiency of 12-mers fragments at different positions in bacterial populations.

[0034] Figure 3b : The number of sequences required to cover more than 99% of bacterial species. The figure shows the difference in efficiency of different segments in covering bacterial species.

[0035] Figure 4 : The number of degenerate bases required when the probability of a single base in each 12-mers reaches 0.99 after 12-mers segmentation; Among them, the number of degenerate bases required when the probability of a single base in each 12-mers sequence reaches 0.99 varies greatly; Overall, the number of degenerate bases required for most segments is relatively stable and remains within a certain range, but the number of degenerate bases increases at some positions, indicating that the diversity of bases at these positions is high, and more degenerate bases may be needed to ensure broad coverage and specificity of the sequence. The introduction of degenerate bases is to cope with the variation in the 16S rRNA sequence of microorganisms and ensure that the primers can cover as many bacterial species as possible; These charts show the changes in different 12-mers fragments after the introduction of degenerate bases, determining the degree of degeneracy required at each position.

[0036] Figure 5 : Examples of alternative primer probe sequences for different detection methods. DETAILED DESCRIPTION

[0037] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0038] Example 1

[0039] This embodiment integrates bioinformatics software tools and 16S rRNA gene sequence data to design a high-coverage PCR primer library that is suitable for different PCR detection technologies (such as TaqMan probes and SYBR Green dye). The 16S rRNA gene refers to the full-length or nearly full-length gene sequence of 16S rRNA of all species of bacteria (Beacteria), with a length between 1400-1500 bases). The design method includes the following steps:

[0040] 1. Data collection and preprocessing

[0041] The full-length or nearly full-length gene sequences of 16S rRNA of all bacterial species (Beacteria) were downloaded from the public database Silva. The downloaded sequences were used to perform quality control on the sequence data to ensure the reliability and accuracy of the data.

[0042] 2. Consensus sequence analysis

[0043] Through the preprocessed 16S rRNA sequence data, by setting the filtering conditions (the ratio of "." and "-" is greater than 0.5 and the base ratio is less than 0.01), we focus on the high-quality and high-consistency regions in the sequence. This helps to intuitively display the conservation and variability distribution of the 16S rRNA gene of all strains in the full length range, and draw a base distribution heat map ( Figure 1a ) and similarity line chart ( Figure 1b ).

[0044] Figure 1a In the figure, highly conserved regions are dark yellow, and the corresponding bases are consistent in most species, indicating that these positions are highly conserved in all bacterial species. Highly variable or missing regions are dark blue or ".", "-" and regions with high frequency of occurrence indicate large variability or deletions, and should not be used for primer design to avoid nonspecific results. Figure 1b A probability line graph of the highest similarity base is shown, with the base and its position in the sequence on the horizontal axis and the probability value on the vertical axis, and a threshold line is drawn at 0.9.

[0045] 3. Screening of highly conserved fragments

[0046] The sequence is divided into continuous 12-mers sequence fragments with a step length of 1 in units of 12 bases. The screening requirements are: in each 12-mers fragment, at least 7 bases have a single base probability greater than 0.99, the median probability of the 12 bases is greater than 0.991, and the base probability of 75% of the sites is greater than 0.995. Using a self-written script, all 12-mers fragments are traversed, and highly conserved fragments that meet the conditions are screened out according to the above standards. After forming a set of candidate sequences, all continuous 12-mers fragments are connected to form a candidate sequence. In this method, 22 candidate sequences of different lengths are formed, which can be further optimized. The screened candidate sequences that meet the conditions are shown in Table 1, and the sequence numbers are SEQ ID NO: 1 to 22:

[0047] Table 1 Candidate primer or probe sequences

[0048]

[0049] The 22 candidate primer sequences obtained by screening were mapped back to the corresponding positions of the bacterial 16S rRNA sequence, and 22 heat maps were generated. Each heat map ( Figure 2a ) shows the distribution of each base in the candidate primer region in different bacterial sequences. In these heat maps, blue indicates a low probability of base occurrence, and yellow indicates a high probability. By analyzing the positions corresponding to the 22 candidate primer sequences, the heat map intuitively shows the distribution of each base in different bacterial 16S rRNA sequences. The bases in the candidate primer region are mainly yellow, indicating the high conservation of these regions. The heat map results show that the positions of the candidate primer sequences are highly conserved and low in variability in the bacterial 16S rRNA gene.

[0050] The similarity analysis of the bacterial 16S rRNA sequence positions corresponding to the candidate sequences was performed, and a similarity line graph was drawn. The horizontal axis represents the specific position of the primer sequence in the 16S rRNA gene and the base with the highest probability, and the vertical axis represents the similarity probability of the bases at these positions. A threshold line was drawn at 0.99 on the vertical axis to distinguish highly conserved regions.

[0051] The line graph shows that the similarity probabilities at most candidate primer positions are above the threshold line of 0.99. This means that these positions are highly conserved in different bacterial species, and the base sequences are almost identical, which makes them suitable for primer design and helps improve the specificity and coverage of primers. The similarity probabilities at a few positions may be slightly lower than 0.99, indicating that there is a certain degree of variability at these positions. These regions need to be carefully considered when designing primers, and degenerate bases may need to be introduced to ensure the effectiveness of the primers. The specific bases and their probabilities are shown in the figure below. Figure 2b shown.

[0052] 4. Introduction and optimization of degenerate bases

[0053] After the screening of highly conserved fragments is completed, the present invention combines two analysis methods to ensure that the primers have high coverage and specificity:

[0054] 4.1 Direct alignment and coverage analysis of 12-mers sequences

[0055] The segmented 12-mers sequences were directly compared with the sequences in the database at the corresponding positions to determine the number of 12-mers sequences required to cover more than 99% of the bacterial sequences to optimize the primer design.

[0056] The candidate sequence was split into 12 bases in length to obtain all possible 12-mers sequence fragments. Each 12-mers sequence was compared with the 16SrRNA sequence in the database using a self-written script, and the coverage of each 12-mers was counted.

[0057] The number of sequences required for each 12-mers fragment to cover more than 99% of bacterial species fluctuated significantly. The overall trend showed that relatively few sequences were required to efficiently cover positions, indicating that the base sequences at these positions were highly conserved in most bacterial species. The sequence coverage efficiency of some fragments was low, indicating that the sequences at these positions were more diverse in bacterial populations, such as Figure 3a Overall, there are significant differences in the conservation and coverage efficiency of 12-mers fragments at different positions among bacterial populations.

[0058] Next, the number of sequences required to cover more than 99% of bacterial species with 22 candidate sequences was counted. The results showed that the number of sequences required to cover more than 99% of bacterial species varied significantly for different sequence segments. Some sequence segments accumulated quickly, and the conservation and coverage of different regions in the database varied greatly. Some sequences were able to quickly reach a coverage threshold of 0.99 when using a small number of sequences, indicating that these segments were highly conserved in bacterial populations, such as Figure 3b shown.

[0059] 4.2 Analysis of single bases and introduction of degenerate bases

[0060] By analyzing the position of each base in the candidate sequence and based on the probability of a single base, the position where a degenerate base needs to be introduced is determined to improve the applicability of the primers to different bacterial species and achieve high coverage.

[0061] Calculate the probability of occurrence of each base position in the candidate sequence, and count the frequencies of the four bases A, T, C, and G at each site. For base positions that do not reach a probability of 0.99, introduce corresponding degenerate bases based on the frequency of base occurrence so that the matching probability of the site reaches or exceeds 0.99. Count the number of degenerate bases and the total number of bases required to make all single bases reach a probability of 0.99 in each candidate sequence.

[0062] Specifically, the number of degenerate bases and the total number of bases required for each single base in the candidate sequence to reach a probability of 0.99 were screened using a self-written script, as shown in Table 2. Then, these candidate sequences were divided into 12-mers units, and the number of degenerate bases required for a single base in each 12-mers sequence to reach a probability of 0.99 was calculated and a line graph was drawn, as shown in Figure 4 Finally, the type and number of degenerate bases that need to be introduced at each base position in the candidate sequence are determined to ensure that the primers achieve high coverage with as little degeneracy as possible. The degenerate bases required to achieve a probability of 0.99 at each base position of the candidate sequence 307-323 are shown in Table 3. Following the same method, the sequences of all 22 candidate sequences after the introduction of degenerate bases are shown in Table 4.

[0063] Table 2 The number of degenerate bases and total number of bases required for each single base in the candidate sequence to reach a probability of 0.99

[0064]

[0065] Table 3 Degenerate bases required to achieve a probability of 0.99 for each base position in candidate sequences 307-323

[0066]

[0067] Table 4 Sequences after introduction of degenerate bases

[0068]

[0069]

[0070] Example 2

[0071] In the examples, the specific operation of searching for microorganisms based on the IUPAC degenerate base coding rules under the SYBR Green dye and TaqMan probe detection system is specifically demonstrated. In order to ensure the wide applicability of this method, especially the verification of the detection rate of four representative microorganisms specified in the General Rules 1101 <Sterility Test Method> of the Fourth Volume of the Chinese Pharmacopoeia, specifically designed primers and probes were used in the experiment. The four microorganisms are:

[0072] Staphylococcus aureus〔CMCC(B)26 003〕

[0073] Pseudomonas aeruginosa〔CMCC(B)10 104〕

[0074] Bacillus subtilis〔CMCC(B)63 501〕

[0075] Clostridium sporogenes〔CMCC(B)64941〕

[0076] (I) Primer retrieval and verification using SYBR Green dye

[0077] Primer sequence design: The forward and reverse primers used in the experiment were designed based on the IUPAC degenerate base rules: Forward Primer (5'-3'): CRAAHVGGMUUAGAKACC (SEQ ID NO: 45);

[0078] Reverse Primer(5'-3'):GGYYGUCGUCAGCUCGUG(SEQ ID NO: 46);

[0079] Among them, the degenerate base R represents A or G; H represents A or U or C; V represents G or Aor C; M represents Aor C; K represents G or U; Y represents C or U;

[0080] This design ensures that the diversity of degenerate bases is also considered in the target sequence to cover the 16S rRNA sequences of different types of bacteria. In this process, a self-written script was used to convert the input degenerate base sequences (such as A, C, G, U, R, Y, S, etc.) into corresponding regular expressions according to the IUPAC coding rules, and automatically match the bacterial 16S rRNA sequences in the database. The script not only retrieves specific sequences, but also verifies them according to the 16S rRNA data of the four bacteria specified in the Chinese Pharmacopoeia. The experimental results show that the primers of SYBR Green dye match the sequences of the above four bacteria, can effectively amplify the target sequence, and can detect and cover 98% of the bacterial sequences, and the detection sensitivity meets expectations.

[0081] (II) Primer and probe design and verification under the TaqMan probe system

[0082] Primer and probe design: Unlike the SYBR Green system, the TaqMan probe system uses the following primers and probes:

[0083] Forward Primer(5'-3'):ACWYCURCGGRDGGCWGCA(SEQ ID NO: 47);

[0084] Reverse Primer(5'-3'):GUGCCASCAGCMGCGGURA(SEQ ID NO: 48);

[0085] Probe (5'-3'): UNAYUGGGYKUAAAGVR (SEQ ID NO: 49); the 5' end of the probe is modified with a fluorescent group HEX (556), and the 3' end is modified with a quenching group BHQ-1.

[0086] Among them, the degenerate bases W represents A or U; Y represents C or U; R represents A or G; D represents G or A or U; S represents G or C; M represents A or C; N represents A or U or C or G; K represents G or U; V represents G or A or C;

[0087] This design ensures the specificity of the probe while taking into account the diversity of degenerate bases to meet the detection needs of different types of microorganisms. Using the IUPAC degenerate base rules, these sequences were converted into regular expressions and searched in the 16S rRNA sequence of the Silva database. According to the script output results, all target bacterial sequences were retrieved. The experimental results show that under the TaqMan probe system, the designed primers and probes successfully matched the four bacteria specified in the General Rules 1101 <Sterility Test Method> of the Fourth Volume of the Chinese Pharmacopoeia.

[0088] After experimental verification, the primer-probe combination using SYBR Green dye and TaqMan probe successfully detected the target microorganisms, effectively amplified the target sequence, and was able to detect 98% of the bacterial sequences, with the detection sensitivity meeting expectations. This set of primers effectively achieves efficient detection of exogenous microbial contamination of cell and gene therapy products, ensuring the accuracy and universality of the detection.

[0089] The above embodiments are only preferred embodiments of the present invention and are not any formal or substantial limitations of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A set of primers, characterized in that The primer is selected from the group consisting of: i) any one or more of the primers whose sequences are shown in SEQ ID NOs: 1 to 22; ii) any one or more of the full-length primers or primer fragments thereof having sequences as shown in SEQ ID NOs: 23 to 44.

2. The method for designing a primer according to claim 1, characterized in that: The following steps are involved: Step 1: Download the full-length or nearly full-length gene sequences of 16S rRNA of all bacterial species (Beacteria) from the public database Silva database, and perform quality control on the downloaded sequence data to ensure the reliability and accuracy of the data; Step 2: Perform consensus sequence analysis on the sequence data obtained in step 1, set filtering conditions (the ratio of ". " and "-" is greater than 0.5 and the base ratio is less than 0.01), and draw a base distribution heat map and a similarity line graph to analyze the highly conserved regions of the sequence; Step 3: Divide the sequence into continuous sequence segments with a step length of 12-mers in units of 12 bases, and select highly conserved segments according to the following screening criteria to form a candidate sequence set, then connect all continuous 12-mers segments to form a candidate sequence, as shown in SEQ ID NO: 1 to 22; The screening criteria are: in each 12-mers fragment, at least 7 bases have a single base probability greater than 0.99, the median probability of 12 bases is greater than 0.991, and 75% of the sites have a base probability greater than 0.

995.

3. The method for designing a primer according to claim 2, wherein: The candidate sequence in step 3 is optimized after introducing degenerate bases to obtain primers with sequences as shown in SEQ ID NOs: 23 to 44.

4. Use of the primer according to claim 1 in the preparation of a reagent or a kit for bacterial detection, wherein the reagent or the kit comprises a primer and / or a probe.

5. A primer, a probe or a primer-probe set for bacterial detection, characterized in that: The primer is selected from any one or more of the full-length primers or primer fragments thereof whose sequences are shown in SEQ ID NOs: 45-46.

6. The primer, probe or primer-probe set according to claim 5, characterized in that: The sequences of the primer probe set are shown in SEQ ID NOs: 47-49, wherein the sequences of the primers are shown in SEQ ID NOs: 47-48, and the sequence of the probe is shown in SEQ ID NO:

49. The 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

7. Use of the primer, probe or primer-probe set according to any one of claims 5 to 6 in the preparation of a reagent or kit for bacterial detection.

8. A reagent or kit for bacterial detection, characterized in that: The reagent or kit comprises a material selected from the group consisting of: i) primers having sequences as shown in SEQ ID NOs: 45-46 and SYBR Green fluorescent dye; ii) a primer-probe set having sequences as shown in SEQ ID NOs: 47 to 49, wherein the sequences of the primers are as shown in SEQ ID NOs: 47 to 48, and the sequence of the probe is as shown in SEQ ID NO: 49.

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