Patent Description:
Short tandem repeats (STRs) are a class of DNA sequences with length polymorphism that are usually formed from <NUM>-<NUM> bases as the core units repeated in tandem in the human genome. The varying number and repeat times of the core units constitutes the genetic polymorphism of STRs. A large amount of STRs are widely distributed throughout the human genome, accounting for approximately <NUM>% of that genome, and thus are highly informative. Various sequences give rise to hundreds of millions of genotype combinations, and each of them occurs at very low frequency in the population and exhibits excellent capability to identify an individual. Moreover, the STR loci with small fragments are more prone to amplification and suitable for detecting trace amounts of tested materials that have been degraded. As each of STR loci can be amplified under similar conditions, they are appropriate for multiplex amplification, which has the advantages of being highly sensitive, accurate, rapid, and hugely informative. Therefore, the STR loci are often used as genetic markers in DNA analytic technology for forensic individual identification and paternity testing.

During the identification of an individual, the source of a sample is affected by the environment and has a very complicated make-up. Typically, a human sample may be contaminated by non-human species or even in some environments interfered apparently by non-human species (such as blood stains), resulting in a sample with lower content or even loss of human-derived DNA. If a sample is detected using only human STR assay kit, circumstances such as lower sample peak, interference with non-specific peak, or even absence of sample peak will be observed so that the person who handles the case cannot immediately determine whether there are too many inhibitors in the sample or whether the DNAs are degraded or present at lower concentrations. It is always required to conduct two or more tests, re-confirm the results on the scene, and determine the source of the sample through further investigation.

Suitable methods that can be employed for identifying non-human species as currently reported in the relevant documents mainly include: <NUM>) the PCR method established by designing species-specific primers based on differences in the mitochondrial DNA (such as cytochrome b (Cytb) gene) sequences (see <CIT>); however, mitochondrial DNAs have high copy number, varying amounts in different tissues, and poor stability, and thus it is difficult to perform quantitative analysis on them; for example, the sequence of Cytb is highly similar among different species and thus is inappropriate for multiplex amplification; moreover, false positive results may arise when the concentration of a sample is high; and <NUM>) Species STR-based detection methods (see <CIT>); however, since STRs are sequences that are highly polymorphic in length, they may occupy a larger portion of the detection range; if combined with detection of human STRs, the multiplex detection quantity for non-human species would be very limited when the detection quantity for human STRs is guaranteed, resulting in lower space utility efficiency within the detection range; if STRs from different species are to be detected jointly in one assay, in practical application they cannot be detected and determined simultaneously by just one test, and multiple amplification and detection are also required. This might be helpful for discriminating purely non-human species to some extent; however, in the case where the human DNAs are contaminated by a non-human sample, the person who conducts the assay would be unable to immediately acquire accurate information about the sample. If there are non-specific amplification peaks for some species in the detection of STRs, the determination of results would be interfered or misinterpreted. Therefore, the assays carried out by such a method are highly restricted and are of little assistance for economizing the resources and improving efficiency.

By performing DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations, Phuchareon et al. (June <NUM>) reveals cross-contamination with cells derived from non-human mammalian species. For routine identification (authentication) of human cell lines, stem cells, and tissues, Nims and al. (<NUM>), propose short tandem repeat (STR) prolfiling. discusses the role of short tandem repeat (STR) profiling within the greater context of cell authentication strategies for reducing the frequency of human and animal cell misidentification. The housekeeping gene ATP5B is known in the art as housekeeping gene for quantitative real-time PCR (del Pozo et al. (<NUM>); Perez-Rico et al. (<NUM>); Turabelidze et al.

In summary, no relevant research or report on a method of detecting human DNA genetic markers for individual identification while simultaneously identifying non-human species has been available yet. To fulfill this need, the present invention provides a detection scheme that is capable of identifying a human-derived sample comprising components of non-human species in a rapid, convenient and accurate manner with high-sensitivity and high-specificity.

The present invention provides a method for rapidly identifying an unknown biological sample suspected to be from human by conducting a joint specific detection of preferable human DNA genetic markers and DNA sequences of housekeeping genes on chromosomes of preferable target non-human species, which can determine in a single assay whether the unknown biological sample is a human-derived sample or a human-derived sample blended with components of other species; if the sample is (or does contain) a human-derived sample, it will be individually identified synchronously. In particular, the present invention provides a multiplex PCR amplification method for species and human individual identification on an unknown biological sample suspected to be from human, which comprises the steps of:.

wherein the premixed PCR reagents comprise primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species, wherein the nuclear gene on the chromosomes of non-human species is housekeeping gene ATP5B; wherein the primers are the primers listed in Tab.

The method for detecting and analyzing PCR amplification products comprises identifying differences in the sizes of PCR product fragments and conducting sequencing analysis on the sequences of PCR products; preferably, the PCR products in the present invention are detected and analyzed by identifying differences in the sizes of the PCR product fragments. Suitable identification methods include polyacrylamide gel electrophoresis, agarose gel electrophoresis and capillary gel electrophoresis. In particular, capillary gel electrophoresis is used in the present invention, which can be divided into single-wavelength and multiple-wavelength methods according to the wavelengths to be detected; preferably, the detection method used herein is a method with <NUM> optical wavelengths.

According to the present invention, the method for individual identification on a human-derived sample is performed by detecting human DNA genetic markers, including InDel sites, SNP sites and STR loci; preferably, <NUM> human STR loci, including D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D2S441, D16S539 and DYS391, as well as a gender recognition site Amel (non-STR locus), are used herein.

The non-human species of the present invention include non-human mammals, such as non-human primates, cats, dogs, sheep, goats, horses, cow, pigs, and rodents; and non-mammals, such as birds, domestic poultry, reptiles, amphibians; preferably, the non-human species used herein are chickens, ducks, geese and pigs.

In the present invention, non-human species are identified by aligning the nucleotide sequences of the same genes among different species and designing species-specific primers and/or probes at locations where the sequences are different to achieve rapid detection and identification of the species. The genes useful for the identification of non-human species may be all the genes that are present on chromosomes of all the species to be identified but are somewhat different in their sequences, such as housekeeping genes in the genome on chromosomes, including genes encoding tubulin, ATP synthase, glycolytic enzymes, ribosomal proteins, etc..

Nuclear genes on chromosomes that are suitable for use in the present invention can ensure that the copy number of nucleic acids in the detected sample is independent of different tissue sources, different life cycles or the like. Preferred housekeeping gene sequences are relatively conservative and stable in genetic evolution, and have significant difference among different species while little difference in the same category of species; they have high species-specificity.

The present invention further provides a multiplex PCR amplification kit for rapidly identifying an unknown biological sample to be detected that is suspected to be from human to determine whether the sample is a human-derived sample or a human-derived sample blended with (or containing) other components of non-human species; if the sample is (or does contain) a human-derived sample, it will be individually identified synchronously. In particular, the kit of the present invention is characterized in comprising primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species, which can be used for synchronous multiple amplification of the following human DNA genetic markers and housekeeping genes on the chromosomes of non-human species: D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D2S441, D16S539, DYS391, and Amel as well as chicken ATP5B, duck ATP5B, goose ATP5B and pig ATP5B.

By performing synchronous PCR amplification of human DNA genetic markers and housekeeping genes from non-human species, the method of the present invention can fulfill joint detection in a single tube for a plurality of species, and further realize synchronous identification of multiple non-human species and various individual identification sites for a human-derived sample. The present method has the advantages of accuracy, high sensitivity, convenience and high specificity, and avoids using multiple PCRs in the current amplification method for identifying origins of species, and thus further avoids wasting resources such as tested samples and testing reagents. When multiple amplifications are not possible due to a small amount of samples, the present method can still succeed in accomplishing the identification.

As described above, the present invention provides a multiplex PCR amplification method for identifying an unknown biological sample suspected to be from human, which comprises the steps of:.

wherein the premixed PCR reagents comprise primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species, species, wherein the nuclear gene on the chromosomes of non-human species is housekeeping gene ATP5B; wherein the primers are the primers listed in Tab.

According to the method described herein, the unknown biological sample refers to a biological sample from which species it is derived is unknown.

In a specific embodiment according to the method of the present invention, the PCR amplification products are detected and analyzed using capillary gel electrophoresis and fluorescence assay. This greatly simplifies the process of the detection and shortens the required time. In a preferred embodiment, the PCR amplification products are detected and analyzed using capillary gel electrophoresis and fluorescence assay with <NUM> optical wavelengths.

The term "DNA genetic marker" as used in the method of the present invention refers to inheritable and detectable DNA sequences that represent the genetic make-up of an organism and are distributed in a population-characteristic pattern.

According to the method of the present invention, the human DNA genetic markers include human STR loci, gender recognition sites, InDel sites, and/or SNP sites.

In one embodiment, the human DNA genetic markers are human STR loci and gender recognition sites; wherein the human STR loci include STR loci on human autosomes and sex chromosomes.

In another specific embodiment, the human DNA genetic markers include the following <NUM> human STR loci: D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D2S441, D16S539 and DYS391, as well as a gender recognition site Amel (non-STR locus).

According to the method of the present invention, the non-human species are non-human mammals, including but not limited to, non-human primates, cats, dogs, sheep, goats, horses, cow, pigs, and rodents (including but not limited to mice and rats); and non-mammals, including but not limited to birds, domestic poultry, reptiles, and amphibians. In a specific embodiment, the non-human species are chickens, ducks, geese and pigs.

According to the method of the present invention, the sample to be detected is selected from blood, blood stains, semen, saliva, body fluids, hair, muscles, or tissues and organs. In some embodiments, the method of the present invention can also be performed directly by amplifying tested materials such as filter paper, FTA cards, cotton pads, and oral swabs.

In the present invention, non-human species are identified by aligning the nucleotide sequences of the same genes among different species and designing species-specific primers and/or probes at locations where the sequences are different to achieve rapid detection and identification of different non-human species. In particular, the genes useful for the identification of non-human species may be all the genes that are present on chromosomes of all the species to be identified but are somewhat different in their nucleotide sequences, such as housekeeping genes on chromosomes, including genes encoding tubulin, ATP synthase, glycolytic enzymes, ribosomal proteins, etc..

The primers of Tab. <NUM> have the following sequences:.

In another specific embodiment according to the method of the present invention, the primers are used at the following concentrations:.

The present invention further provides a multiplex PCR amplification kit for identifying species and human individuals on an unknown biological sample suspected to be from human, which is characterized in comprising primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species, species, wherein the nuclear gene on the chromosomes of non-human species is housekeeping gene ATP5B; wherein the primers are the primers listed in Tab. In some embodiments, the human DNA genetic markers include human STR loci, gender recognition sites, InDel sites and/or SNP sites; preferably, the human DNA genetic markers are human STR loci and gender recognition sites.

In some embodiments, the nuclear genes on the chromosomes of non-human species include all the genes that are present on chromosomes of all the species to be identified but are somewhat different in their nucleotide sequences. Preferably, the nuclear genes are housekeeping genes on chromosomes; more preferably, the housekeeping genes include genes encoding tubulin, ATP synthase, glycolytic enzymes, and/or ribosomal proteins.

In a specific embodiment, the kit comprises primers for amplifying the following human DNA genetic markers and housekeeping genes on the chromosomes of non-human species: D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D2S441, D16S539, DYS391, and Amel as well as chicken ATP5B, duck ATP5B, goose ATP5B and pig ATP5B.

The primers listed in Tab. <NUM> have the following sequences:.

In another specific embodiment, the primers are used at the following concentrations:.

In addition to the primers listed above, the kit of the present invention further comprises nuclease-free water, PCR reaction mixture, primer mixture, allele mixture, and internal standard SIZE-<NUM> Plus as components. It is worth mentioning that the PCR reaction mixture as used herein has been subject to a series of optimization experiments so as to afford a product that is compatible with all the ordinary tested sample materials available on the Chinese market, including whatman FTA cards, whatman saliva cards, blood filter paper, Bokun FTA cards, Bokun saliva cards, hair, exfoliated cells from the oral cavity, extracted DNAs and the like. Moreover, such an improved buffer can greatly increase the amplification efficiency, effectively shorten the time for terminal adenylation of products and the overall amplification duration, and improve the amplification efficiency of longer fragments and the uniformity of the product. The PCR reaction mixture comprises the following main components: DMSO, Tris-buffer, potassium chloride, ammonium sulfate, dNTP, Tween <NUM> and betaine.

The present invention will be further described in detail in the following examples in conjunction with the accompanying drawings.

In the practical identification application, contamination by non-human species is primarily caused by ordinary animals in urban and rural areas. Thus, four ordinary non-human species (namely, chickens, ducks, geese and pigs) that are readily available are selected for use in the present application. Sequences of the same gene in each species are selected from the database of NCBI as the detected sequence. By aligning these sequences, a specific region on ATP5B is selected and experimentally verified.

In the present invention, the following <NUM> human chromosomal STR loci are determined by screening: D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D2S441, D16S539 and DYS391, as well as a gender recognition site Amel (non-STR locus); wherein DYS391, a core site in the China National GeneBank DataBase and a gender recognition site located on chromosome Y, is used for auxiliary sex determination.

Six-color fluorescent labels (namely, blue, green, yellow, red, purple, and orange labels) were selected for use in the present invention after discriminating and screening fluorescent dyes to construct a combined scheme of six-color fluorescence. When the combined scheme of six-color fluorescence had been determined, the combination of loci and types of the fluorescent labels were designed through numerous repeated experiments. Based on the production costs and the amplification efficiency of the primers for each locus, the above loci were divided into five groups:.

Specific primers were first designed at regions flanking repeated sequences of the <NUM> loci described above using Primer Premier5 software. The annealing temperature for each primer was around <NUM>. No primer dimers, other interactions or cross-reactions would be present. The amplification product has a length ranging from <NUM> to 500bp. Each pair of primers was assayed for amplification and optimized until a clear single band was observed after amplification.

Secondly, as to non-human species, ATP5B gene was selected by searching the gene library, which expresses beta polypeptide of F1 complex of ATP synthase. Specific portion of DNA in the gene was selected by aligning the DNA sequences of the same gene from different species using DNAMAN. Specific primers were designed using the Primer Premier5. No primer dimer, other interactions or cross-reactions would be present. After amplification, there was a clear single band for the target species. Multiplex amplification was optimized and validated for different species. No non-specific amplification peaks for other species than the target species, especially no interference peaks for pure human DNA, would occur.

Each locus was separated by the lengths of the fragments amplified by the primers in each of the above groups; and sequences of the primer were optimized so that no non-specific band was present within the amplification range to ensure high-efficient detection. Simultaneously, detection was performed on different species to ensure high species specificity. Subsequently, the concentrations of the primers were adjusted such that accurate STR typing on human samples and specific detection on non-human species are possible at <NUM> ng of positive control samples from both human and non-human species; and the heights of amplification peak of the same color remained <NUM>% or higher at equal amounts of the DNAs of the human and non-human species. Sequences and concentrations of the specific primers used are shown in Table <NUM> below:.

The multiplex PCR amplification kit for identifying species and human individuals on an unknown biological sample suspected to be from human as provided herein comprises:.

The above PCR Master comprises: <NUM>-<NUM> DMSO, <NUM>-<NUM> Tris-buffer, <NUM>-<NUM> potassium chloride, <NUM>-<NUM> ammonium sulfate, <NUM>-<NUM> deoxynucleotide triphosphate (dATP, dGTP, dTTP, dCTP), <NUM>-<NUM>/mL BSA, <NUM>-<NUM>% Tween <NUM>, and <NUM>-<NUM> betaine, and is compatible with various testing materials that have been commonly used for amplification and available on the market.

The above Primer Mix comprises all the primers for amplifying <NUM> detection sites (their concentrations are shown in Table <NUM>), <NUM>-4U/25µl-reaction Taq polymerase, <NUM>-<NUM> magnesium chloride, etc..

The above Control DNA 9948A is human genomic DNA, purchased from Suzhou Xinhai Biotechnology Co.

The above Allelic Ladder allelic genotyping standard is a collection of the distribution of each allele having all the different genotypes at the above-mentioned loci in certain numbers of populations.

The above SIZE-500Plus orange fluorescent molecular weight internal standard is a series of amplification products used to calibrate certain fragment sizes.

The above spectral calibration standards are fluorescent PCR amplification products of <NUM> fragments having different sizes.

Configuration of the reaction system.

Experimental protocol for thermocycler amplification.

<NUM>) Placing the PCR amplification tube on the thermocycler;
<NUM>) Performing the amplification by selecting the procedures as recommended below;
<NUM>) Keeping the amplified samples away from light.

Fluorescence assay of the amplification products on genetic analyzer.

A loading mixture (<NUM>-<NUM>µL SIZE-<NUM> Plus + <NUM>µL deionized formamide) was prepared from deionized formamide and the molecular weight internal standard (SIZE-<NUM> Plus) in the system. <NUM>µL of the loading mixture was blended with <NUM>µL of the amplification product or allelic genotyping standard in the system (Allelic ladder). Air bubbles should be avoided and electrophoresis was carried out as soon as possible. The amplification product was detected and analyzed using a genetic analyzer (such as ABI <NUM>/<NUM> series). The data obtained after electrophoresis were analyzed on GeneMapper ID-X data analysis software to obtain genotyping spectrum and data.

Sensitivity analysis: After the positive control was diluted according to certain copy number fold, it was assayed by PCR amplification and capillary electrophoresis until no signal was detected. This copy number was considered as the lowest detection limit, i.e., the sensitivity of the kit. The highest sensitivity refers to the sensitivity when DNA samples at a concentration as low as <NUM> ng can be detected.

Specificity analysis: Control DNA <NUM>, chicken, duck, goose, pig, dog, sheep, mouse, cow, E. coli and the like were assayed with fluorescently-labeled multiplex amplification and validation system at <NUM> tested sites according to the present invention. For the Control DNA <NUM>, only human STR loci were observed; for the chicken, duck, goose, and pig, only the corresponding specific peaks were observed; and for the rest of the species, no specific amplification peak was observed, indicating that this system has high specificity for the identification of species.

The kit provided by the method of the present invention is used for detecting samples from unknown sources. The detection steps are described as follows:.

The results show that the sample from unknown source contains a small amount of human DNA and is also blended with a large amount of porcine DNA.

Claim 1:
A multiplex PCR amplification method for identifying an unknown biological sample suspected to be from human, which can determine in a single assay whether the unknown biological sample is a human-derived sample or a human-derived sample blended with components of other species; if the sample is (or does contain) a human-derived sample, it will be individually identified synchronously; and the method comprises the steps of:
<NUM>) in an unknown biological sample that has been collected, wherein the sample is suspected to be from human;
<NUM>) adding the unknown biological sample to be detected directly, or the nucleic acids extracted from the unknown biological sample to be detected, into premixed PCR reagents;
<NUM>) performing multiplex PCR amplification by running PCR amplification procedures;
<NUM>) detecting and analyzing the PCR amplification products;
wherein the premixed PCR reagents comprise primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species;
wherein the nuclear gene on the chromosomes of non-human species is housekeeping gene ATP5B;
wherein the primers comprised in the premixed PCR reagents have the following sequences:
Amel: SEQ ID NO: <NUM>-<NUM>;
D8S1179: SEQ ID NO: <NUM>-<NUM>;
D21S11: SEQ ID NO: <NUM>-<NUM>;
D18S51: SEQ ID NO: <NUM>-<NUM>;
D2S1338: SEQ ID NO: <NUM>-<NUM>;
Pig ATP5B: SEQ ID NO: <NUM>-<NUM>;
DuckATP5B: SEQ ID NO: <NUM>-<NUM>;
D2S441: SEQ ID NO: <NUM>-<NUM>;
D5S818: SEQ ID NO: <NUM>-<NUM>;
D7S820: SEQ ID NO: <NUM>-<NUM>;
D6S1043: SEQ ID NO: <NUM>-<NUM>;
Penta D: SEQ ID NO: <NUM>-<NUM>;
Goose ATP5B: SEQ ID NO: <NUM>-<NUM>;
D3S1358: SEQ ID NO: <NUM>-<NUM>;
TH01: SEQ ID NO: <NUM>-<NUM>;
D19S433: SEQ ID NO: <NUM>-<NUM>;
D12S391: SEQ ID NO: <NUM>-<NUM>;
Chicken ATP5B: SEQ ID NO: <NUM>-<NUM>;
DYS391: SEQ ID NO: <NUM>-<NUM>;
TPOX: SEQ ID NO: <NUM>-<NUM>;
D16S539: SEQ ID NO: <NUM>-<NUM>;
D13S317: SEQ ID NO: <NUM>-<NUM>;
FGA: SEQ ID NO: <NUM>-<NUM>;
CSF 1PO: SEQ ID NO: <NUM>-<NUM>;
vWA: SEQ ID NO: <NUM>-<NUM>;
D1S1656: SEQ ID NO: <NUM>-<NUM>;
Penta E: SEQ ID NO: <NUM>-<NUM>.