**3. Result and discussion**

The four sets of designed primer pairs were submitted to specificity using Primer-Blast and the results revealed that, they are specific to amplify the 16S rRNA gene of humans (*Homo sapiens*), sheep (*Ovis aries*), goats (*Capra hircus*), and cows

(*Bos taurus*) (**Table 2**). The 16S rDNA region is a highly conserved region among mtDNA [17]. mtDNA can be easier to retrieve from low-quantity and/or degraded DNA samples, as it is present at many copies per cell, thus providing a clear advantage over nuclear genome-based methods of species identification [18–20].

The results of PCR testing revealed that the primer pairs were specific and nonspecific products did not appear for all samples. The amplification of Homo sapiens mtDNA with primer pairs of other (Ovis aries, Capra hircus, and Bos taurus) and amplification of each with primer pairs of another genus gave negative results. This was primary evidence for primer pair specificity. The amplicon of 16S rRNA gene of Homo sapiens was 1200 bp (**Figure 1A**), Ovis aries was 1060 bp (**Figure 1B**), Capra hircus was 820 bp (**Figure 1C**), and Bos taurus was 1300 bp (**Figure 1D**). PCR amplification and sequence analysis of the mitochondrial 16S rRNA gene was utilized for differentiation/identification and subsequently evaluation of their application in


**27**

**Figure 1.**

*Mitochondrial 16S rRNA Gene-Dependent Blood Typing as a Forensic Tool*

solving forensic cases [21]. Mitochondrial 16S is suitable for the differentiation of 300 mammalian species. The 16S rDNA gene is a common mitochondrial gene for detection of blend mutton and pork at high sensitivity. The mitochondrial 16S rRNA genes have been used as molecular markers to identify mammals, birds, shrimps, and other species using species-specific primers that amplify the 12S rRNA or 16S rRNA gene regions from mtDNA [17, 22]. Gene loci on the mitochondrial genome have been used in species identification. These include the 12S and 16S rRNA loci. The D-loop (displacement loop) has been used less in species identification but more in intraspecies identification. Due to the greater sequence variation at this non-coding locus, it is now being used as a tool for identifying the presence of particular species

*of Bos taurus 16S rRNA gene. Lanes H1–H18 represent samples. M represents 100 bp DNA ladder.*

*Agarose gel electrophoresis 1.5% for: (A) 1200 bp amplicon of Homo sapiens 16S rRNA gene. Lanes H1–H18 represent samples. (B) 1060 bp amplicon of Ovis aries 16S rRNA gene. Lanes H1–H18 represent samples. (C) 820 bp amplicon of Capra hircus 16S rRNA gene. Lanes H1–H18 represent samples. (D) 1300 bp amplicon* 

The secondary and confirmatory assay for specificity of primer pairs used in the study was sequences of PCR products. Eight amplicons from each were sent for sequencing using the Sanger technique (Macrogen/Korea). The retrieved sequences firstly must be trimmed to remove unwanted sequences before submitting them for BLASTN. The trimming performed by Bioedit was utilized to obtain the finally processed sequences. Abbreviations of Homo sapiens sequences were used as (HIS-1 to HIS-8), Ovis aries sequences be (IOA-1–IOA-8), Capra hircus sequences

The identity percentage and alignment results of the amplified 16S rRNA gene of Homo sapiens, Ovis aries, Capra hircus, and Bos taurus with database were

*DOI: http://dx.doi.org/10.5772/intechopen.98248*

within mixture of many species [23, 24].

illustrated in **Tables 3**–**6**, respectively.

(IBCH-1–IBCH-8), and Bos taurus sequences (IBT-1–IBT-8).

**Table 2.** *Primer-blast of designed primer pairs.* *Mitochondrial 16S rRNA Gene-Dependent Blood Typing as a Forensic Tool DOI: http://dx.doi.org/10.5772/intechopen.98248*

#### **Figure 1.**

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

tage over nuclear genome-based methods of species identification [18–20].

(*Bos taurus*) (**Table 2**). The 16S rDNA region is a highly conserved region among mtDNA [17]. mtDNA can be easier to retrieve from low-quantity and/or degraded DNA samples, as it is present at many copies per cell, thus providing a clear advan-

The results of PCR testing revealed that the primer pairs were specific and nonspecific products did not appear for all samples. The amplification of Homo sapiens mtDNA with primer pairs of other (Ovis aries, Capra hircus, and Bos taurus) and amplification of each with primer pairs of another genus gave negative results. This was primary evidence for primer pair specificity. The amplicon of 16S rRNA gene of Homo sapiens was 1200 bp (**Figure 1A**), Ovis aries was 1060 bp (**Figure 1B**), Capra hircus was 820 bp (**Figure 1C**), and Bos taurus was 1300 bp (**Figure 1D**). PCR amplification and sequence analysis of the mitochondrial 16S rRNA gene was utilized for differentiation/identification and subsequently evaluation of their application in

**Gene Primer sequence 5**′ **to 3**′ **Sequence ID of isolate Identity**

MN115376.1 MN053904.1 MN125706.1 MN163828.1 MN163832.1 MN125705.1 MN163282.1 MN125704.1 MN124446.1 MK069579.1

KP998473.1 KP998472.1 KP998470.1 KP702285.1 MH841968.1 MH841967.1 MH841966.1 MG837554.1 MG837553.1 KU681224.1

LS992662.1 LS992661.1 LS992659.1 LS992658.1 LS992656.1 LS992655.1 LS992654.1 LS992653.1 LS992652.1 LS992651.1

EU177866.1 EU177865.1 EU177864.1 EU177863.1 EU177862.1 EU177861.1 EU177860.1 EU177859.1 EU177858.1 EU177856.1

100%

100%

100%

100%

F:GCCTGGTGATAGCTGGTTGT R:ATCATTTACGGGGGAAGGCG

F:AGGCCTAAAAGCAGCCATCA R:GCCCTTTTCTAGGGCAGGTT

F:GCCTGGTGATAGCTGGTTGT R:TCACCCCAACCAAAACTGCT

R:GGGCAGGGTTTTGTGTTGTC

*Bos taurus* 16S rRNA F:CTAAGCAGCCCGAAACCAGA

**26**

**Table 2.**

*Primer-blast of designed primer pairs.*

*Homo sapiens* 16S

rRNA

*Ovis aries* 16S rRNA

*Capra hircus* 16S

rRNA

*Agarose gel electrophoresis 1.5% for: (A) 1200 bp amplicon of Homo sapiens 16S rRNA gene. Lanes H1–H18 represent samples. (B) 1060 bp amplicon of Ovis aries 16S rRNA gene. Lanes H1–H18 represent samples. (C) 820 bp amplicon of Capra hircus 16S rRNA gene. Lanes H1–H18 represent samples. (D) 1300 bp amplicon of Bos taurus 16S rRNA gene. Lanes H1–H18 represent samples. M represents 100 bp DNA ladder.*

solving forensic cases [21]. Mitochondrial 16S is suitable for the differentiation of 300 mammalian species. The 16S rDNA gene is a common mitochondrial gene for detection of blend mutton and pork at high sensitivity. The mitochondrial 16S rRNA genes have been used as molecular markers to identify mammals, birds, shrimps, and other species using species-specific primers that amplify the 12S rRNA or 16S rRNA gene regions from mtDNA [17, 22]. Gene loci on the mitochondrial genome have been used in species identification. These include the 12S and 16S rRNA loci. The D-loop (displacement loop) has been used less in species identification but more in intraspecies identification. Due to the greater sequence variation at this non-coding locus, it is now being used as a tool for identifying the presence of particular species within mixture of many species [23, 24].

The secondary and confirmatory assay for specificity of primer pairs used in the study was sequences of PCR products. Eight amplicons from each were sent for sequencing using the Sanger technique (Macrogen/Korea). The retrieved sequences firstly must be trimmed to remove unwanted sequences before submitting them for BLASTN. The trimming performed by Bioedit was utilized to obtain the finally processed sequences. Abbreviations of Homo sapiens sequences were used as (HIS-1 to HIS-8), Ovis aries sequences be (IOA-1–IOA-8), Capra hircus sequences (IBCH-1–IBCH-8), and Bos taurus sequences (IBT-1–IBT-8).

The identity percentage and alignment results of the amplified 16S rRNA gene of Homo sapiens, Ovis aries, Capra hircus, and Bos taurus with database were illustrated in **Tables 3**–**6**, respectively.


**Table 3.**

*Identity of blasted isolates (IHS-1–IHS-8) with reference sequences of highest identity percentage.*


**Table 4.** *Identity of blasted isolates (IOA-1–IOA-8) with reference sequences of highest identity percentage.*


#### **Table 5.**

*Identity of blasted isolates (IBCH-1–IBCH-8) with reference sequences of highest identity percentage.*

The sequencing of the 16S rRNA has revolutionized the study and identification of human and non-human samples in forensic science. A simple method was developed using universal primers for species identification based on direct PCR sequencing using primer sets that were designed based on the conserved regions of the 16S rRNA loci detected by the comprehensive sequence comparison among 30 animals whole [25]. The mitochondrial DNA method could be a dominant tool for mammalian species identification, especially in forensic cases in which many unidentified biological samples need to be analyzed such as blood spots [25].

**29**

verification.

**Table 6.**

real-time PCR.

**4. Conclusions**

*Mitochondrial 16S rRNA Gene-Dependent Blood Typing as a Forensic Tool*

The 16S and 12S sequences allow identification of most species to the genus level. Faster-evolving DNA regions are required to identify closely related animal species [26]. The successfully used forensically informative nucleotide sequencing analysis of the 16S rRNA mitochondrial DNA were very valuable to identify before unknown biological specimens of human and animals [27]. The mitochondrial 12S rRNA and 16S rRNA genes, including those from fish and amphibians to mammals including human beings. Therefore, universal primers were designed to amplify sequences in the fast-evolving animal mtDNA [17]. The PCR amplifications of mitochondrial 16S rRNA followed by sequencing and analysis were demonstrated to be very efficient for identification of species origin [21]. The 12S rRNA and 16S rRNA gene sequences of animals reveal the fitting level of interspecific variation but the great level of intraspecific homogeneity [7]. The results showed no crossreactivity of designed primer pairs and the PCR assay based on the designed primer

*Identity of blasted isolates (IBT-1–IBT-8) with reference sequences of highest identity percentage.*

**Isolate Sequence ID Expect Identities Gaps Strand** IBT-1 MF169214.1 0.0 99.50% 3/601(0%) Plus/Plus IBT-2 KT184455.1 7e-139 100.00% 0/273(0%) Plus/Plus IBT-3 KT184466.1 0.0 99.32% 7/1177(0%) Plus/Plus IBT-4 KT184466.1 0.0 99.90% 0/979(0%) Plus/Plus IBT-5 KT184466.1 0.0 99.90% 0/965(0%) Plus/Plus IBT-6 KT184466.1 0.0 100.00% 0/512(0%) Plus/Plus IBT-7 KT184466.1 0.0 99.81% 1/1077(0%) Plus/Plus IBT-8 KT184466.1 0.0 100.00% 0/1092(0%) Plus/Plus

The extraction of mtDNA and amplification of mtDNA genes seem to be accessible, very easy, and cheap. Additionally when the sequences of amplified genes analyzed the results were very clear and no confusion with other genes. The accuracy is very high due to no cross-amplification between species-specific primers that were observed. The sensitivity is also high due to that the mixed blood at a very small amount (10%) can be detected. Their strength over another technique like real-time PCR was, post real time, cannot perform the sequencing when needed for

The only weakness in the technique is requirement for more time, preparation, possibility of contamination, and more machine when compared with

There is sensitivity, specificity, and accuracy of the designed species-specific primer pairs and applicability of the designed primer pairs in forensics to investi-

gate blood spots or evidence belonging for human, sheep, goat, and cow.

pairs will be simple, fast, sensitive, specific, and cost-effective.

**3.1 Strength of PCR-dependent 16S mtDNA gene**

**3.2 Weakness of PCR-dependent 16S mtDNA gene**

*DOI: http://dx.doi.org/10.5772/intechopen.98248*

*Mitochondrial 16S rRNA Gene-Dependent Blood Typing as a Forensic Tool DOI: http://dx.doi.org/10.5772/intechopen.98248*


**Table 6.**

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

**Isolate Sequence ID Expect Identities Gaps Strand** IHS-1 MH444415.1 0.0 98.98% 0/885(0%) Plus/Plus IHS-2 MK069579.1 0.0 99.74% 0/771(0%) Plus/Plus IHS-3 MK069579.1 0.0 99.57% 2/697(0%) Plus/Plus IHS-4 MK069579.1 0.0 99.08% 0/654(0%) Plus/Plus IHS-5 MK059695.1 0.0 99.86% 0/701(0%) Plus/Plus IHS-6 MK069579.1 0.0 99.39% 0/657(0%) Plus/Plus IHS-7 MK295855.1 0.0 99.50% 0/599(0%) Plus/Plus IHS-8 MK069579.1 0.0 99.69% 0/653(0%) Plus/Plus

**Isolate Sequence ID Expect Identities Gaps Strand** IOA-1 MG489885.1 0.0 98.70% 0/769(0%) Plus/Plus IOA-2 MG489885.1 4e-170 100.00% 0/329(0%) Plus/Plus IOA-3 MG489885.1 0.0 98.98% 0/586(0%) Plus/Plus IOA-4 MG489885.1 0.0 99.73% 0/749(0%) Plus/Plus IOA-5 MG489885.1 0.0 99.63% 0/542(0%) Plus/Plus IOA-6 MG489885.1 2e-157 99.36% 0/312(0%) Plus/Plus IOA-7 MG489885.1 0.0 99.17% 2/483(0%) Plus/Plus IOA-8 MG489885.1 0.0 99.80% 0/489(0%) Plus/Plus

*Identity of blasted isolates (IHS-1–IHS-8) with reference sequences of highest identity percentage.*

*Identity of blasted isolates (IOA-1–IOA-8) with reference sequences of highest identity percentage.*

**Isolate Sequence ID Expect Identities Gaps Strand** IBCH-1 KP271023.1 0.0 99.02% 0/614(0%) Plus/Plus IBCH-2 KP271023.1 0.0 98.58% 0/633(0%) Plus/Plus IBCH-3 KP271023.1 2e-174 99.74% 0/378(0%) Plus/Plus IBCH-4 KP271023.1 0.0 100.00% 0/729(0%) Plus/Plus IBCH-5 KP271023.1 0.0 99.83% 0/595(0%) Plus/Plus IBCH-6 KP271023.1 0.0 100.00% 0/480(0%) Plus/Plus IBCH-7 KP271023.1 0.0 99.84% 0/618(0%) Plus/Plus IBCH-8 KP271023.1 0.0 99.17% 0/481(0%) Plus/Plus

The sequencing of the 16S rRNA has revolutionized the study and identification of human and non-human samples in forensic science. A simple method was developed using universal primers for species identification based on direct PCR sequencing using primer sets that were designed based on the conserved regions of the 16S rRNA loci detected by the comprehensive sequence comparison among 30 animals whole [25]. The mitochondrial DNA method could be a dominant tool for mammalian species identification, especially in forensic cases in which many unidentified biological samples need to be analyzed such as blood spots [25].

*Identity of blasted isolates (IBCH-1–IBCH-8) with reference sequences of highest identity percentage.*

**28**

**Table 4.**

**Table 5.**

**Table 3.**

*Identity of blasted isolates (IBT-1–IBT-8) with reference sequences of highest identity percentage.*

The 16S and 12S sequences allow identification of most species to the genus level. Faster-evolving DNA regions are required to identify closely related animal species [26]. The successfully used forensically informative nucleotide sequencing analysis of the 16S rRNA mitochondrial DNA were very valuable to identify before unknown biological specimens of human and animals [27]. The mitochondrial 12S rRNA and 16S rRNA genes, including those from fish and amphibians to mammals including human beings. Therefore, universal primers were designed to amplify sequences in the fast-evolving animal mtDNA [17]. The PCR amplifications of mitochondrial 16S rRNA followed by sequencing and analysis were demonstrated to be very efficient for identification of species origin [21]. The 12S rRNA and 16S rRNA gene sequences of animals reveal the fitting level of interspecific variation but the great level of intraspecific homogeneity [7]. The results showed no crossreactivity of designed primer pairs and the PCR assay based on the designed primer pairs will be simple, fast, sensitive, specific, and cost-effective.

#### **3.1 Strength of PCR-dependent 16S mtDNA gene**

The extraction of mtDNA and amplification of mtDNA genes seem to be accessible, very easy, and cheap. Additionally when the sequences of amplified genes analyzed the results were very clear and no confusion with other genes. The accuracy is very high due to no cross-amplification between species-specific primers that were observed. The sensitivity is also high due to that the mixed blood at a very small amount (10%) can be detected. Their strength over another technique like real-time PCR was, post real time, cannot perform the sequencing when needed for verification.

#### **3.2 Weakness of PCR-dependent 16S mtDNA gene**

The only weakness in the technique is requirement for more time, preparation, possibility of contamination, and more machine when compared with real-time PCR.

#### **4. Conclusions**

There is sensitivity, specificity, and accuracy of the designed species-specific primer pairs and applicability of the designed primer pairs in forensics to investigate blood spots or evidence belonging for human, sheep, goat, and cow.
