TW201335375A - Method for improving sensitivity and specificity of screening assays for KRAS codons 12 and 13 mutations - Google Patents

Abstract

A method of diagnosing a KRAS gene mutation at codons 12-13 in a DNA sample is disclosed. The method comprises detecting one or more than one mutation in the KRAS gene codons 12-13 of the DNA sample by performing an allelic discrimination assay using a mutant probe, a wild-type probe paired with the mutant probe, a forward primer and a reverse primer, the mutant probe being adapted to detect a single nucleotide mutation at 1A, 1T, 1C, 2A, 2T, 2C or 5A of the KRAS gene codons 12-13 of the DNA sample, and the primers each having no greater than 25 nucleotides in length are adapted to amplify a region spanning KRAS exon 2 codons 12-13, wherein the mutant and wild-type probes are labeled with different fluorescent dyes.

Description

Method for improving the sensitivity and specificity of detecting KRAS code 12 and 13 mutations

The present invention relates to a method for detecting a mutant of the KRAS c12-13 gene, which can specifically and sensitively diagnose cancer and/or evaluate the prognosis of cancer.

KRAS (official name: v-Ki-ras2 Kirsten mouse sarcoma virus homologous oncogene; alias: KRAS2, RASK2; gene bank accession number NM_033360, all incorporated into the present invention for reference) is a type of activation in EGFR Small GTPases in the message transmission pathway affect the growth, survival and differentiation of cancer cells. EGFR is thought to be a carcinogen that causes abnormal proliferation of epithelial cells, and anti-EGFR drugs have a deteriorating effect on millions of cancer patients worldwide. The constituent proteins produced by the C12-13 mutant K-ras, the GTPase is continuously activated, and the GTP linkage is also altered. The K-ras mutant protein downstream of EGFR, the persistently activated GTPase, will make the anti-EGFR drug efficacy less effective.

Errors and substitutions of single nucleotides in the K-ras gene occur mainly at the 1st, 2nd, and 5th nucleotide positions encoding 12 and 13 (>98%), common in pancreatic cancer (75-90) %), lung adenocarcinoma (20-50%), and colon cancer (CRC, 30-60%) and other epithelial tumors, while patients with K-ras mutations are not recommended to use anti-EGFR drugs, such as panitumumab and cetuximab ( VECTIBIX® and ERBITUX®) are blocked due to treatment effects. KRAS detection has become a guideline for the treatment of lung adenocarcinoma and CRC patients, and the assessment of KRAS mutation status can also provide predictive values for cancer progression, metastasis, and CRC genetic potential. Detection of KRAS mutations in the plasma of patients with pancreatic cancer can be used to determine the residual of tumor cells, which is helpful to evaluate the effect of treatment in patients; detection of KRAS mutations in bile also contributes to the early diagnosis of biliary tract cancer.

At present, the rapid screening of KRAS hotspot mutations is developing positively. The detection effect depends on the combination of probes and primers. However, how the design and combination can improve the sensitivity and specificity of allele identification. Less discussed.

In one aspect, the invention relates to a method for detecting a 12-13 mutation of a KRAS gene in a DNA sample, the method comprising: a mutant probe labeled with different fluorescence, and a wild type probe corresponding thereto Needle, with a forward and a reverse primer for allele identification. The above-mentioned mutant probes are directed to major single nucleotide mutations located at 12-13 of the KRAS gene, including: 1A, 1T, 1C, 2A, 2T, 2C or 5A, with a pair of no more than 25 nucleotides each. The length of the primer is amplified across the segment of KRAS exon 2 coding 12-13, wherein the fluorescent markers of the mutant and wild-type probes are each Fam and Vic.

Other aspects and modifications may be made without departing from the spirit and scope of the invention.

The drawings and the description of the present invention are intended to be illustrative of the scope of the invention, and the same reference numerals are used to represent the same or similar elements in the embodiments.

Each of the terms used in the context of the invention, as well as in a particular context, substantially have its ordinary meaning in the art to which it pertains. The specific terms used to describe the invention are discussed below or elsewhere in the specification for further guidance. The practitioner is referred to the description of the invention. For convenience, specific terms in the specification will be emphasized by, for example, italics and/or quotation marks, and this emphasis does not affect the scope and meaning of the term, and in the same context, with or without special emphasis, The term has the same meaning and meaning. In addition, since the same matter may be described in a variety of ways, any one or more of the terms discussed herein may also be replaced by a word or a synonym, but not any of the specific meanings of the specification are discussed and detailed herein. The present invention provides synonym for specific terms, but one or more of the synonyms listed herein does not exclude the use of other synonyms. Any term used anywhere in this specification is for illustrative purposes only and is not intended to The scope and meaning of the invention or any exemplary term may be limited. Likewise, the various embodiments of the present specification are not intended to limit the scope of the invention.

Unless otherwise defined, all technical and scientific terms related to the present invention have the same meaning as those skilled in the art, and if there are differences, they will be defined in the specification.

As used herein, "around", "about" or "approximately" means a given value or a percentage of a given range of 20, preferably within a percentage of 10, more preferably as a percentage. Within 5. Here, if the given quantity does not specifically indicate "around" or "about" Or the term "approximately" can also presume that the given quantity implies an approximate meaning.

It is to be understood by those skilled in the art that the <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Here, "a segment spanning the KRAS exon 2 coder 12-13" means "a nucleotide sequence comprising the sequence 12-13 of the KRAS gene exon 2 coder".

Herein, the term "paired probes" or "a pair of probes" means a probe pair comprising a mutant probe and a wild type probe, wherein the wild type The design of the probe pair paired with the mutant probe.

The quality control (QC) mutant is the same DNA sequence as the mutant probe. The amount of QC mutant used was fixed to 1 to 5% of the amount of QC wild-type DNA in parallel PCR. For example, when the mutant probe 2CM (SEQ ID NO: 5) is used, the QC mutant needs to comprise the nucleotide sequence of SEQ ID NO: 5, and when the mutant probe NIA is used (SEQ ID NO: 9) In case, the QC mutant type needs to include the nucleotide sequence of SEQ ID NO: 9 and the like.

Herein, the term "comparable intensity" means that the fluorescence intensity of the DNA sample reaches a fluorescence intensity equivalent to that of the QC mutant, indicating that the DNA sample has the mutant type. When detecting DNA samples, parallel detection of QC mutants is the establishment of a baseline fluorescent reading for mutation identification.

A single base pairing error reduces the energy and melting point (Tm) of the base bond and is the basis for the hybridization of allele specific oligonucleotides (ASO). The application of ASO is common in traditional dot blot, real-time PCR (qPCR) fluorescent probes and the current microarray DNA wafers, and the strength of the bonding structure is determined by the Tm of the probe sequence and the opening and expansion. The difficulty of the secondary structure of the amplicon. Therefore, the sequence of the probe and the length or structure of the amplicon play an important role in clinical testing based on ASO.

The present invention uses real-time PCR (qPCR) as a platform to simultaneously enhance and detect the c12-13 segment of K-ras, and uses an primer capable of amplifying the segment and a hybridization probe capable of identifying a mutant to perform an oligonucleotide sequence. Specific allele detection. In a preferred embodiment, the small groove junction (MGB) of the Taqman probe enables short probes (13-20 nucleotide strands) to have a high melting point difference (ΔTm). The present invention utilizes allele specific oligonucleotide (ASO) hybridization on a fast, simple qPCR to improve K-ras c12-13 by the sequence and pairing of probe pairs and primer pairs. Detection ability of mutant detection.

Identification of the K-ras genotype by qPCR must distinguish the mutant alleles at each point in the majority of the wild type. There are two levels of this difficulty: one is the heterogeneity of the test material (the gene for the test sample) itself, and the limit of the test method. The heterogeneity of the test gene may be mixed by the tumor to the non-tumor component template, the wild type and the mutant DNA are unevenly distributed, and the mutation is heterozygous or homozygosity. Recently, K-ras mutation detection mostly uses primary tumor tissue (primary The tumor tissue) is not only easy to obtain, but also has a sufficient amount of tumor cells. The endoscopic lens is used to identify the tumor before surgery, and the distribution of cancer cells can also be confirmed by the slice. About 20% of patients with metastatic cancer have not previously found a primary tumor, ie no tumor removal and sections are preserved. Therefore, the test is performed only by a sample of a metastatic tumor tissue that has just been removed, such as a liver or a positive lymph node. However, it is not uncommon for the K-ras status to be inconsistent in primary and metastatic tumor tissues. (Mariani et al. (2010) "Concordant analysis of KRAS status in primary colon carcinoma and matched metastasis. Anticancer Res" 30, 4229-35)

Different probes have different affinities for secondary structures of amplicons (especially in the vicinity of binding motifs), and it is not possible to improve allele probes and primers only by melting point (Tm) estimation. . First, search for suitable primers (such as AS12F and AS12R) and partial probe sequences using ABI's SNP software v1.3, and evaluate them by parallel testing and replacement of individual factors (such as probes, primers, Taq DNA polymerase). As a result, after selecting a better factor, it was confirmed three times. Reference Chow, L. et al. (2012) "Differences in the frequencies of K-ras c12 by allelic detection by pathological phenotypes of sex and colon cancer -13 genotypes by gender and pathologic phenotypes in colorectal tumors measured using the allele discrimination method. Environmental and Molecular Mutagenesis)" 53: 22-31, also incorporated herein by reference. In addition, different sample sources (tissue sections, fresh or frozen tissue, patient serum), the type of template (gDNA or cDNA) contained, and the amount used are also evaluated to confirm the effect.

The Taq DNA polymerase herein is not particularly limited and may include TaqMan Fast Universal PCR Master Mix (Applied Biosystems, CA, USA) and KAPA probe rapid qPCR kit (KAPA Probe Fast qPCR). Kit) (Kapa Biosystems, MA, USA).

The qPCR instrument may include ABI 7900 and ABI 7500 Fast, but is not limited thereto.

Detection application

Human DNA contains genomic DNA (gDNA) and complementary DNA (cDNA) for reverse transcription of mRNA. In one embodiment of the invention, the paraffin-embedded (FFPE) human tissue gDNA line is extracted with the DNeasy Blood & Tissue Kit (Qiagene, Valencia, CA, USA). On the other hand, mRNA of frozen tissues or freshly cultured cancer cell lines can be extracted and reverse transcribed into cDNA using Trizol (Invitrogen, NY, USA). The invention can simultaneously detect seven alleles in the sample, and each time the detection reaction is no matter how many samples (in the 8x12 well plate, the necessary QC mutant type is removed, the maximum is 11 samples), all must contain different Mutant QC to interpret the detection results. All seven reactions were identical except for the allele-specific probe.

For clinical applications, the present invention utilizes a suitable extraction solution to extract rich and fresh serum DNA as a sample.

In one aspect, the invention relates to a method for detecting a single nucleotide mutation in the 12th and 13th coding sequences of KRAS in a DNA sample, comprising: by using a mutant probe, a corresponding wild type Probe, a forward and a reverse primer, using a allelic discrimination method to detect whether a DNA sample has One or more mutations in KRASc12-13. The mutant probe is directed against a 1A, 1T, 1C, 2A, 2T, 2C or 5A single nucleotide mutation on KRASc12-13, and amplifies the primer of the 12-13 segment of the KRAS exon 2 coder, both of which are No more than 25 nucleotides; mutant and wild-type probes are labeled with different fluorescent dyes.

The mutant and wild type probe pairings are selected from the group consisting of: (i) SEQ ID NO: 1 paired with SEQ ID NO: 2; (ii) SEQ ID NO: 3 paired with SEQ ID NO: 4; SEQ ID NO: 5 is paired with SEQ ID NO: 2; (iv) SEQ ID NO: 7 is paired with SEQ ID NO: 8; (v) SEQ ID NO: 9 is paired with SEQ ID NO: 8 or 10; SEQ ID NO: 11 is paired with SEQ ID NO: 10; and (vii) SEQ ID NO: 12 is paired with SEQ ID NO: 13.

In an embodiment of the invention, the detecting step comprises: (a) mixing the DNA sample with the paired probe, the primer, and the polymerase chain reaction (PCR) reagent; (b) amplifying the crossover KRAS exon 2 coder Sections 12-13; (c) measuring the fluorescence intensity of the mutant probe in the DNA sample; and (d) comparing the fluorescence intensity of the corresponding quality control (QC) mutant, if comparable or stronger It means that it has a mutant type.

In another embodiment of the invention, the forward and reverse primers each independently comprise the nucleotide sequences SEQ ID NO: 14 and 15.

In another embodiment of the present invention, the method comprises: detecting a mutant of 1A, 1T, 1C, 2A, 2T, 2C, and 5A on KRASc12-13, wherein the probe pairing comprises the following paired sequence: (i SEQ ID NO: 1 is paired with SEQ ID NO: 2; (ii) SEQ ID NO: 3 is paired with SEQ ID NO: 4; (iii) SEQ ID NO: 5 is paired with SEQ ID NO: 2; (iv) SEQ ID NO:7 is paired with SEQ ID NO:8; (v) SEQ ID NO:9 is paired with SEQ ID NO:8 or 10; (vi) SEQ ID NO:11 is paired with SEQ ID NO:10; and (vii) SEQ ID NO: 12 is paired with SEQ ID NO: 13.

In another embodiment of the invention, the mutant probe is labeled with FAM.

In another embodiment of the invention, the wild type probe is labeled with VIC.

In another embodiment of the invention, each probe is no more than 18 or 16 nucleotides in length.

In another embodiment of the invention, the primer is used to amplify an amplicon of less than 80 or 70 base pairs (bp).

In another embodiment of the invention, the primer is used to amplify a DNA fragment comprising the nucleotide sequence of SEQ ID NO: 16.

In another embodiment of the invention, the primer is used to amplify a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 16.

In another embodiment of the invention, the step of amplifying comprises performing an instant PCR (qPCR).

In another embodiment of the invention, the DNA sample comprises a tumor biopsy (biopsy), paraffin embedded (FFPE) tumor tissue section, fresh or Frozen DNA tissue, or gDNA or cDNA prepared from cultured cancer cell lines.

In another embodiment of the invention, the section is from a mucinous or metastatic rectal cancer, or a cholangiocarcinoma.

In another embodiment of the invention, the cDNA is prepared in frozen tumor tissue.

In another embodiment of the invention, the DNA is obtained from the serum of a CRC patient.

In another embodiment of the present invention, the amount of gDNA is not more than 10 ng / μl.

Example

Without limiting the scope of the invention, the instrument, apparatus, method and results of the embodiments of the invention are provided below. The names and subtitles used in the examples to facilitate the reader do not limit the scope of the invention, and the theory presented and disclosed herein, whether correct or not, falls within the scope of the invention, and regardless of any theory or function. Combinations, as long as the invention can be practiced, are within the scope of the invention.

Example 1 Sample collection and analysis of alleles of 7 KRAS mutants

The gDNA of 21 tumor cell lines (colon cancer or lung cancer) was subjected to KRAS sequencing and testing, and the tumor samples were collected from 204 volunteer patients who participated in the colon cancer from 2007 to 2009. All tumor samples were treated with hematoxylin. Hematoxylin and Eosin staining was re-evaluated by pathologists from the Cancer Institute of the National Institutes of Health of Taiwan. The study was approved by the Human Body Testing Committee of the Chengdu University Hospital.

Synchronous (Y) and wild type were detected by synchronizing the 1A, 1T, 1C, 2A, 2T, 2C and 5A mutant probes indicated by Fam and the wild-type probes labeled with Vic (as designed in Table 1). (X) allele. For each real-time PCR (qPCR) reaction, add 1X TaqMan Genotyping Master Mix (P/N4352042, available from Applied Biosystem) or 1X KAPA Probe Fast qPCR Kit Master Mix Universal (KK4701, Purchased from Kapa Biosystems), 20 or 10 ng of gDNA or 100 ng of cDNA was reverse transcribed from RNA, 3.38 pmole of Fam- and Vic-labeled probe pairs, and 9 pmole of primers. The procedure for setting the ABI PRISM 7900HT or 7500 Fast Sequence Detection System (available from Applied Biosystems) was: 2 minutes at 50 ° C, 10 minutes at 95 ° C, then 15 cycles after 15 seconds at 95 ° C. 60 seconds at 60 ° C. The QC mutant of the positive control group is produced by site directed mutagenesis, and the DNA plastid obtained after extensive replication is mixed with 1-5% in the wild-type DNA plastid. Each test includes a no-template control (NTC) as a negative control.

Fluorescent labeling probes for all K-ras mutants detected by all alleles are listed in Table 1.

In Table 1, probes 1TM, 2TM, 2CM, and 5AM detect mutations of 1T, 2T, 2C, and 5A, respectively, while new probes N1A, N2A, and N1C detect single mutations of the 1A, 2A, and 1C bottom lines, respectively.

PCR primer: The sequence of the introduced primer was as follows: forward primer: 1F : TGACTGAATATAAACTTGTGGTAGTTG (SEQ ID NO: 14); AS12F: AGGCCTGCTGAAAATGACTGAATAT (SEQ ID NO: 21; constructed from the exon 2 front-end sequence).

Reverse primer: AS12R : GCTGTATCGTCAAGGCACTCTT (SEQ ID NO: 15); 1R: TCgTCCACAAAATgATTCTgAA (SEQ ID NO: 22; constructed from the exon 2 rear-end sequence).

A 66 bp amplicon was synthesized by a primer (bottom line). The wild type amplicon sequence is: TGACTGAATATAAACTTGTGGTAGTTG GAGCT GGTGGC GTAGGC AAGAGTGCCTTGACGATACAGC (SEQ ID NO: 16), where the bottom line is 1F and AS12R (complementarity) )the sequence of.

On the other hand, the sequence of the mutant amplicon that may be synthesized by 1F and AS12R can be expressed as: tgactgaa tataaacttg tggtagttgg agct NNtgAc gtaggcaaga gtgccttgac gatacagc, the letter "NN" represents three possible nucleotides other than wild type G (A, C, T).

Binding to the 1F and AS12R primers paired with the new K-ras probe (the sequence title indicated by the bold in Table 1) can improve the allelic detection of the seven common mutants of K-ras c12-13.

The sequence of exon 2 of K-Ras NM_004985 is as follows: Tggtagttgg agct ggtggc gtaggcaaga gtgccttgac gatacagcta Ccaacaatag ag/(SEQ ID NO: 17), the coding 12 segment is ggt , the coding 13 region is ggc , the framed sequence is AS12F and 1R (complementary sequence), and the SEQ ID NO: 17 sequence comprises SEQ ID NO: 16 sequence.

In a total volume of 15 μl of the PRC reaction, the final concentrations of the components are as follows:

Table 3 lists the location and type of single nucleotide mutations detected.

data analysis:

The Fam intensity (Y) of each allele reaction in the sample is compared with the QC mutant corresponding to 1% to 5%, which is the distinguishing point of this qualitative measurement (Figures 2 and 3), except for the pure mutant Outside (homogeneous mutant), representing the wild type of Vic (X-axis), either strong or weak will be seen in all samples. The signal presentation is output by the excel file whether it is a color pattern or a number. Where the Y value of the sample is equal to or higher than the QC mutant Y value, it is determined to have a mutation. The bar graph in the examples represents the average of the Y values obtained for multiple experiments.

Although the allelic discrimination test is a qualitative analysis method, by increasing the difference in Fam intensity from the wild type (ΔY), the measured amount of the QC mutant is increased from the initial 5% to the last less than 1%. The process of improving this assay involves a change: pairing of primers to shorten the length of the amplicon from 80 to 66 bp, and the sequence of the probe and its pairing (Example 4). Finally, probes N1A, N1C containing primers 1F and AS12R, 1T, N2A, 2C, 2T and 5A have been shown to achieve sensitivity in detecting 1% QC mutants.

The invention proves that the allele-specific probe and the primer sequence and the length of the amplicon may affect the detection effect, and the changed sequence is further improved by the combination of different reagents, instruments and sample types, and further improves the detection sensitivity and specificity. Sexual space.

Example 2 Compare RFLP and sequence sensitivity and specificity

To compare the results of the three methods, sensitivity was defined as the proportion of measurements that were confirmed to be correct mutations (considered as a positive control), while the specificity measures were confirmed to be the correct proportion of wild type (considered as a negative control). If the allelic result measured by one method is different from the other two methods, it is regarded as a false positive or a false negative.

(positive possibility for correct positive)

(Possibility of negative for correct negative)

21 strains of cancer cells were initially tested by allele detection, sequencing and RFLP methods, and six mutations with K-ras c12 were confirmed (A549 and H358 were in the first base and SW480, SW620, PRMI-8226 and H2444 were in the second base). Base), eight mutations with c13 (H1355, H1734 and H1755 in the first base and DLD-1, HCT-8, HCT-116, Lovo and SW48 in the second base), and seven K-ras wild type. One of the 15 mutant cells detected by RFLP is The pseudo-mutant type (6.7%) (A172) was caused by incomplete restriction of the cleavage of the nucleic acid by the restriction enzyme, and this problem was worsened in the FFPE tissue (n=62) (Table 4).

Of the 25 K-ras mutants detected by RFLP, 8 were confirmed to be pseudo-mutations (8/25=32%) by allele detection and sequencing, 16 correct and 1 with different mutations. Type (1/17 = 6% false mutant). Since RFLP is poor in the sensitivity of FFPE samples (>10% or more of mutants can be detected in mixed samples) and specificity (82%, Table 4), it is replaced by dHPLC as the third test to confirm the allele. The difference between the results of the gene and the sequencing test.

The sequencing method has a 12.4% error rate in FFPE tissue samples, and there are many noises in the pattern of the sequencing results. Some of the samples show poor PCR yield before sequencing. The first time sequence is a failure test, and the test is sent again after at least double the amount. The study found that DNA sequencing detected more than the allele method with double mutants (Table 6), and most of them were false by the third method, thus slightly reducing the sensitivity and specificity of the DNA sequencing method ( Each is 98.8% and 97.5%, Table 5). When sequencing some older tissues, it was found that biased mutations vary with the type of Taq polymerase (Viogene, Qiagene, Invitrogen, and KB HotStart), possibly due to biodegradation or poor tissue processing. Fragmentation. Newer DNA sequencing techniques such as pyrosequencing and next generation sequencing (NGS) are more likely to overcome the above problems without going through the PCR step.

RFLP detection is often unpredictable due to PCR manipulation and restriction enzymes (as shown in Table 4), which is also time consuming (at least two days). In addition to the result of 12.4% error rate, the overall sequence still has high sensitivity (98.8%, Table 5) and specificity (97.5%), which is suitable for clinical testing; but its technical limitations and errors The high rate leads to difficulties in troubleshooting and delays in results. Table 4 shows the RFLP results for 62 CRC samples, and Table 5 compares the results of the allele and DNA sequencing assays.

RFLP sensitivity = 100% (17/17) and specificity = 82% (37/45).

The pseudo MT of RFLP = 32% (8/25) and the wrong MT = 6% (1/17).

The correctness of the RFLP results was confirmed by the results of sequencing and allelic detection.

Example 3 Other verification of allelic detection

The third assay (dHPLC) has confirmed that the sensitivity (100%) and specificity (100%) of the allelic assay are higher than the sequencing, and the genotype results are compared in Table 6. Among the different genotypes measured by the allelic assay and the sequencing method, one mutant (5A) and three wild types (sequenced as one wild type, one 2T, 2A5A and 1C5A) confirmed by dHPLC were correct. Double mutation). Although double mutations are associated with intratumoral genetic heterogeneity within the tumor, they require microdissection to confirm. Compared to the qPCR method, the double mutation tends to occur in sequencing and SSCP assays [Bazan et al., 2002; Span M, 1996]. Overall, the detection of 204 CRC samples resulted in 83 K-RAS mutations (40.7%), of which 20% were G12D (GAT), 7.4% were 12V (GTT), and 7.4% were 13D (GAC), and 5.3% of the sum of the other four mutations (12C, 12R, 12A and 12S).

The two 2A5A and 1C5A (bottom line) double mutations in the sequence were determined to be wild-type by the allelic method (dHPLC confirmed), and the other two mutations were also sequenced. The two double mutations -2A5A, 2T5A and 1A2A - were detected separately. For single mutations 2A, 2T and 2A; in addition, the sequenced single mutation 2C, in a mucinous histotype, was detected as a double mutation 2C2A. * In a metastatic lung cancer, sequencing and allelic detection simultaneously detected the double mutation 1A2A. ~ Deduct the repetition of double mutations.

The accuracy of the allelic detection method is divided into two types. The intra-run precision is the random test of the repeatability of 21 samples, and the inter-run precision evaluation. Five samples were taken five times together with the repeatability of each QC group (5% QC mutant, WT and NTC), of which five CRC samples were two 2A mutants (P18 and P48), one 2T mutant (P30) and two wild types (P02 and P04) (Chow et al., 2012). The standard deviation (SDs) of the Fam fluorescence (MT) values of each sample was small in three or five tests, confirming that the accuracy and reproducibility of the test were good. Therefore, by comparing the results of different methods and statistical analysis of the test in patients The accuracy and reproducibility of the sample, the allelic test does meet the requirements of clinical testing (CLIA guidelines).

In a small number of cholangiacarcinoma samples (n=40), the KRAS mutation results of allele detection and DNA sequencing were evaluated and compared. It was found that the allele detection method detected more than the sequencing method. Two mutants and one different mutant, after deducting 13 tumor cells with less than 5% of the samples, at least 7% of the detection rate. The performance of the test depends on monitoring the necessary QC, which is used to distinguish between the occurrence of each error before the test (sample and preparation) or the test itself, and to monitor the stability of the test operation (Figure 1).

Example 4 Allele detection by improved new oligonucleotide (ASO)

Unlike RFLP and DNA sequencing, which undergo secondary PCR amplification, allele detection can simultaneously amplify and detect genotypes, reducing the probability of PCR amplification errors. After the new primer shortens the length of the qPCR amplicon (from 80 bp to 66 bp), the detection rate and sensitivity of the FFPE sample are improved. On the contrary, the sequence amplicon must be a fragment of 150 to 250 bp. The new primer 1F replaces AS12F with the original primer AS12R, and the new probe (N1A, N1C, N2A and N2C in Table 1) greatly improves the detection sensitivity by detecting 5% mutant (Fig. 1) to 1%. Mutant (Figures 5 and 6). The new N2A (Fig. 2A) and N2C (Fig. 3A) probes are allele-detected, and the older (A, circled dots) distinguish 5 to 100% of the 2A or 2C mutants. From the difference in Fam(Y) intensity between mutant and wild type (△Y=Y _MT -Y _WT ), it can be seen that the sensitivity of the new primer 1F to increase the N2A probe is increased from 5% of the detected 2A mutant to 1% (Fig. 2B). However, the 1F primer is not necessarily superior to the old 2C probe for the new N2C probe (Fig. 3B). The old 2C probe is shown when the QC mutant of 5% 2C and 2C5A is detected and the RPMI-8226 cells containing 2C are detected. The ΔY of the needle is preferred. Therefore, except for 2C, the ΔY of all detected 5% mutants is shown in Figure 4A, and the Fam(Y) value of the N1C probe was monitored for two months (Fig. 4B).

Example 5 Performance of assays in different Taq polymerase and qPCR instruments

Compare the different reagents with the new ASO sequence, the Kapa Probe Fast qPCR Kit and the TaqMan Fast Universal PCR Master Mix (Figure 5), as well as the instrument, ABI 7900 and 7500 Fast (Figures 6A and 6B). The new ASO sequence was tested with 1% and 2% QC mutants, and the measured Fam intensity (Y) as a percentage increases reflects sensitivity.

Example 6 Reduce the amount of gDNA required for assays and possible applications of assays

The amount of gDNA in each reaction was reduced from 20 to 10 ng (Figure 7). The combination of the new ASO detected the K-ras genotype of the two co-cultured cells, and the 10 ng gDNA (Fig. 8) and 100 ng cDNA (Fig. 9) corresponded to the K-ras genotype of individual cells. When analyzing the RNA of a few CRC patients with frozen tissue and a small amount of serum samples, although the 2G and 5A mutants were detected in the tumor gDNA, 6 serum DNAs frozen at -80 °C for more than 1 year were not detected (Fig. 10). The 2T and 2A double mutants were detected by reverse transcription of cDNA into frozen tissue, but the validation of gDNA data was lacking. Although limited in sample size, K-ras detection is a non-invasive diagnostic tool for circulating DNA and tissues. The potential of RNA may also have a large clinical impact, such as rapid screening of immediate genetic variations in high-risk individuals. The invention is simple and rapid, and is a detection method with great clinical application value.

The above-described embodiments are for illustrative purposes only and are not intended to be exhaustive or limiting.

The described embodiments and examples are intended to be illustrative of the principles of the invention and the application of the embodiments of the invention. The skilled artisan will clearly convert the embodiments without departing from the spirit and scope of the invention, and the scope of the invention is defined by the appended claims rather than the foregoing description or embodiments.

The patents, patent applications, and various references, which are hereby incorporated by reference in their entirety, are hereby incorporated by reference in their entirety in their entirety in their entirety herein All references cited and discussed in this specification are all identified by the same principles, as if each reference has its own referenced.

Figure 1 shows monthly monitoring of the K-ras allele assay in the quality control group (QC) containing wild-type (wt) and 5% mutant (mt) plastid DNA, which was performed during the course of serving CRC ( Chow et al.)

Figure 2 shows the sensitivity of the K-ras assay, measured by measuring 5% 2A mutant (A: the lowest percentage in the distinguishable range) to 1% (B), a new mutation Type probe - N2A (CTGATGGCGTAGGC; SEQ ID NO: 11) with wild type probe TTGGAGCTGGTGGC (SEQ ID NO: 10), plus new primer 1F (TGACTGAATATAAACTTGTGGTAGTTG; SEQ ID NO: 14) with primer AS12R (GCTGTATCGTCAAGGCACTCTT; SEQ ID NO: 15) achieved. However, the ability to resolve mutants with the primer pair 1F/1R (TCgTCCACAAAATgATTCTgAA; SEQ ID NO: 22) or AS12F (AGGCCTGCTGAAAATGACTGAATAT; SEQ ID NO: 21)/AS12R, N2A mutant probe was unstable.

Figure 3A shows that the N2C mutant probe (CC TACGCCAGCAGC; SEQ ID NO: 6) initially appeared to have a similar N2A trend with the wild type probe (CCTACGCCACCAGCT; SEQ ID NO: 2), but further observed the difference between 5% 2C and 2C5A plastid DNA and Y difference (ΔY) (B) of 2C-containing RPMI-8226 cell genomic DNA (gDNA), with the same 1F primer (TGACTGAATATAAACTTGTGGTAGTTG; SEQ ID NO: 14) with AS12R (GCTGTATCGTCAAGGCACTCTT; SEQ ID NO: 15) was found to be slightly inferior to the old 2C probe (CTACGCCAGCAGCT; SEQ ID NO: 5). Whether the new or old probe distinguishes the 2C allele, the effect with AS12F (AGGCCTGCTGAAAATGACTGAATAT; SEQ ID NO: 21) / AS12R primer pairs is better than 1F (SEQ ID NO: 14) / 1R ( TCgTCCACAAAATgATTCTgAA; SEQ ID NO: 22) primer pair.

Figure 4 shows that in addition to 2C, the new probe and the primer detect ΔY (the sensitivity at the time) of all 5% QC mutants; among them, the N1C mutant probe (TTGGAGCTCGTGGC; SEQ ID NO: 12) The ability to distinguish alleles with the wild type probe TTGGAGCTGGTGGCGT (SEQ ID NO: 13) appears to be better (monitoring in June and July 2009) (B).

Figure 5 shows the sensitivity of the alleles to detect 1% and 2% QC mutants with new probes and primers (in terms of ΔY), ABI reagent (solid bar graph) - TaqMan rapid universal PCR premix reagent (TaqMan) Fast Universal PCR Master Mix) is superior to the cheaper KAPA probe fast qPCR kit (KAPA Probe Fast qPCR Kit) (hollow strip diagram) (A); but in the new probes 1A, 1C and 2A ( Within the circle), KAPA reagent also has good sensitivity (B).

Figure 6 compares the sensitivity of the new probes and primers to detect 1% and 2% QC mutants (in terms of ΔY) and the differences between ABI 7900 (A) and 7500 (B) instruments.

Figure 7 compares the effects of 10 and 20 ng of cellular gDNA on K-ras allele detection (new primers and probes) with comparable results. 1C was omitted because cells having the 1C mutant type were not found, and the detailed probe pair is shown in Table 1.

Figure 8 shows that the new probe and primer detect cell gDNA 10 ng. The K-ras genotype obtained from the co-cultured two cells corresponds to individual genotypes. For example, wild-type Honel co-cultured with 5A-derived DLD1. (H+D) 5A was measured; the respective 2T, 1A and 5A probe pairs are shown in Table 1.

Figure 9 shows that the new probe and primer detect 100 ng of cell cDNA. The genotypes obtained from the two cells co-culture correspond to individual genotypes, for example, SW480 with 2T and DLD1 co-culture with 5A (S+D) 2T and 5A were measured; 1A of A549 was weakened when co-cultured with Honel or DLD1, but not with SW480, and it was not excluded that gene expression may be disturbed during co-culture.

Figure 10 shows 6 CRC patients with no detectable mutations in serum DNA (20 ng) (five intact samples), tumors of patients I20 and I350 (T; non-tumor, N) gDNA To the 2T and 5A mutants; in addition, the frozen tumor tissue cDNA (100 ng) of the patient I78 (in the right small table) was found to have a 2A2T double mutant, but the lack of gDNA was tested.

<110> National Institute of Health Zhou Yihui Liu Wei

<120> Method for improving the sensitivity and specificity of detecting KRAS code 12 and 13 mutations (METHODS FOR IMPROVING SENSITIVITY AND SPECIFICITY OF SCREENING ASSAYS OF KRAS CODONS 12 AND 13 MUTATIONS)

<130> S 3525/0556-P 040TW

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<170> PatentIn version 3.5

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Claims (20)

A method for detecting a mutant of a KRAS gene codon 12-13 in a DNA sample, comprising: using a mutation in an allelic detection method in the KRAS gene coding 12-13 of the DNA sample a probe, a wild type probe paired with the mutant probe, a forward primer and a reverse primer to detect a mutant of the KRAS gene coding 12-13 in the DNA sample, wherein the mutant probe A single nucleotide mutation in the 1st, 1T, 1C, 2A, 2T, 2C or 5A of the KRAS coder 12-13 in a DNA sample is detected, and each primer is not more than 25 nucleotides in length to enlarge the horizontal A segment spanning a KRAS exon 2 coding fragment 12-13, wherein the mutant and wild-type probes are labeled with different fluorescent dyes. The method of claim 1, wherein the detecting step comprises: (i) mixing the DNA sample with the mutant and the wild type probe pair, a primer pair, and a polymerase chain reaction (PCR) reagent; (ii) Amplifying the segment spanning the KRAS exon 2 coder 12-13; (iii) measuring the intensity of the fluorescent dye of the mutant probe in the DNA sample; and (iv) comparing the mutant The fluorescence intensity of the probe and its corresponding quality control (QC) mutant is representative of the presence of the mutation if it is comparable or strong. The method of claim 1, wherein the forward and the reverse primer comprise nucleotide sequences SEQ ID NO: 14 and 15, respectively. The method of claim 1, wherein the mutant and the wild type probe are a probe pair selected from the group consisting of (i) SEQ ID NO: 1 paired with SEQ ID NO: 2; (ii) SEQ ID NO: 3 paired with SEQ ID NO: 4; (iii) SEQ ID NO: 5 paired with SEQ ID NO: 2; SEQ ID NO: 7 is paired with SEQ ID NO: 8; (v) SEQ ID NO: 9 is paired with SEQ ID NO: 8 or 10; (vi) SEQ ID NO: 11 is paired with SEQ ID NO: 10; Vii) SEQ ID NO: 12 is paired with SEQ ID NO: 13. The method of claim 4, wherein the forward and the reverse primer are the nucleotide sequences SEQ ID NO: 14 and 15, respectively. The method of claim 1, wherein the mutant probe is labeled with FAM. The method of claim 6, wherein the wild type probe is labeled with VIC. The method of claim 1, comprising detecting a mutation of 1A, 1T, 1C, 2A, 2T, 2C and 5A on KRAS codon 12-13, the probe pair comprising the following: (i) SEQ ID NO : 1 paired with SEQ ID NO: 2; (ii) paired with SEQ ID NO: 3 and SEQ ID NO: 4; (iii) paired with SEQ ID NO: 5 and SEQ ID NO: 2; (iv) SEQ ID NO: 7 is paired with SEQ ID NO: 8; (v) paired with SEQ ID NO: 9 and SEQ ID NO: 8 or 10; (vi) paired with SEQ ID NO: 11 and SEQ ID NO: 10; Vii) Pairing of SEQ ID NO: 12 with SEQ ID NO: 13. The method of claim 8, wherein the forward and reverse primers are nucleotide sequences SEQ ID NO: 14 and 15, respectively. The method of claim 8, wherein the detecting step comprises: (a) mixing the DNA sample with the mutant and the wild type probe pair, a primer pair, and a polymerase chain reaction (PCR) reagent; Amplifying the segment spanning the KRAS exon 2 coder 12-13; (c) measuring the intensity of the fluorescent dye of the mutant probe in the DNA sample; and (d) comparing the respective The fluorescence intensity of the mutant probe and its corresponding quality control (QC) mutant, if it is comparable or strong, represents the presence of the mutation. The method of claim 1, wherein each probe does not exceed 18 or 16 nucleotides in length. The method of claim 1, wherein the DNA sample comprises: a tumor biopsy sample, a paraffin-embedded tumor tissue section (FFPE), a fresh or frozen tumor mass, or a cultured cancer cell. A genetic DNA (gDNA) or cDNA prepared by the strain. The method of claim 12, wherein the tumor section is from a colorectal cancer (CRC), a mucinous or metastatic tumor, a cholangiocarcinoma or a lung cancer. The method of claim 12, wherein the cDNA is prepared from a frozen or fresh tumor tissue. The method of claim 2, wherein the step of amplifying comprises performing a real time PCR or qPCR. The method of claim 1, wherein the primer is suitable for amplifying an amplicon of less than 80 or 70 base pairs (bp). The method of claim 15, wherein the primer is adapted to amplify a DNA fragment comprising the nucleotide sequence of SEQ ID NO: 16. The method of claim 15, wherein the primer is adapted to amplify a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 16. The method of claim 1, wherein the DNA sample comprises serum DNA obtained from one of CRC patients. The method of claim 12, wherein the amount of the sample gDNA does not exceed 10 ng/μl.