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. 2021 Dec 20;41(12):1816-1821.
doi: 10.12122/j.issn.1673-4254.2021.12.09.

[Combined detection of Helicobacter pylori 16S rRNA and cagA gene in saliva specimens using multiplex PCR]

[Article in Chinese]
X Wang  1   2 , L Sun  1 , Z Yang  3 , S Song  1 , N Li  1 , Y Liu  1 , W Tian  3 , Y Zhao  1
Affiliations

[Combined detection of Helicobacter pylori 16S rRNA and cagA gene in saliva specimens using multiplex PCR]

[Article in Chinese]
X Wang et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To establish a multiplex PCR-based method for detecting Helicobacter pylori (Hp) 16S rRNA gene and cagA gene in saliva samples for investigating the prevalence of Hp in the oral cavity of Hp-infected patients with digestive tract diseases.

Methods: Bioinformatics technique was used to design specific primers for Hp 16S rRNA and cagA genes for Hp detection using multiplex PCR, with recombinant cloning plasmids serving as the standard positive control. Oral saliva samples were collected from 156 patients with digestive tract diseases, and Hp 16S rRNA and cagA genes were detected using the established multiplex PCR system.

Results: The established multiplex PCR system showed a strong specificity and a high sensitivity for detecting Hp 16S rRNA gene and cagA gene, with the lowest detection limit of 103 copies/μL. The recombinant plasmids pGMT-16s and pGMT-cagA could be used as standard positive controls for the identification of Hp. Among the 156 saliva samples, 87.2% were positive for Hp 16S rRNA gene and 23.1% for Hp cagA gene.

Conclusion: Hp is highly prevalent in saliva specimens of Hp-infected patients with digestive tract diseases. The presence of Hp in the oral cavity may importantly contribute to Hp infection in the digestive tract and recurrence after treatment.

目的: 检测唾液标本幽门螺杆菌（Hp）的16S rRNA基因及cagA基因，了解消化道疾病患者口腔中Hp存在情况及致病情况。

方法: 利用生物学信息技术，设计Hp 16SrRNA、cagA基因特异性引物，建立检测Hp 16SrRNA、cagA基因的多重PCR方法；重组克隆质粒构建标准阳性对照品；收集156例消化道疾病患者的唾液标本，提取唾液标本中细菌DNA，应用建立的多重PCR方法，检测Hp 16SrRNA基因及cagA基因，并对结果进行分析。

结果: 建立的检测Hp 16SrRNA基因及cagA基因的多重PCR方法特异性强，灵敏性较高，最低检测线为103 copies·μL-1；重组质粒pGMT-16s和pGMT-cagA，可作为鉴定Hp的标准阳性对照品；检测Hp感染的消化道疾病患者的唾液标本，口腔携带Hp 16SrRNA基因的阳性率为87.2%，cagA基因阳性率为23.1%。

结论: Hp感染的消化道疾病患者口腔唾液标本中存在Hp，口腔中存在Hp可能是诱发消化道Hp感染及治疗后复发的一个重要原因。

Keywords: 16S rRNA gene; Helicobacter pylori; cagA gene; saliva.

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Figures

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引物Hp-16s特异性试验 Primer Hp-16s specificity test. M: D2000 DNA Marker; N: Blank; Lane 1: Hp standard strain; Lane 2: Escherichia coli; Lane 3: Staphylococcus aureus; Lane 4: Campylobacter rectus; Lane 5: Streptococcus salivarius; Lane 6: Lactobacillus.
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引物Hp-cagA特异性试验 Primer Hp-cagA specificity test. M: D2000 DNA Marker; N Blank; Lane 1: Hp standard strain; Lane 2: Escherichia coli; Lane 3: Staphylococcus aureus; Lane 4: Campylobacter rectus; Lane 5: Streptococcus salivarius; Lane 6: Lactobacillus.
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引物Hp-16s灵敏性试验 Primer Hp-16s sensitivity test. M: D2000 DNA Marker; N Blank; Lane 1: 100 ng·μL-1; Lane 2: 10 ng·μL-1; Lane 3: 1 ng·μL-1; Lane 4: 1×10-1 ng·μL-1; Lane 5: 1×10-2 ng·μL-1; Lane 6: 1×10-3 ng·μL-1; Lane 7: 1×10-4 ng·μL-1.
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引物Hp-CagA灵敏性试验 Primer Hp-CagA sensitivity test. M: D2000 DNA Marker; N: Blank; Lane 1: 100 ng·μL- 1; Lane 2: 10 ng·μL-1; Lane 3: 1 ng·μL-1; Lane 4: 1×10-1 ng·μL-1; Lane 5: 1×10-2 ng·μL-1; Lane 6: 1×10-3 ng·μL-1; Lane 7: 1×10-4 ng·μL-1.
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多重PCR凝胶电泳图谱 Multiplex PCR gel electrophoresis pattern. M: D2000 DNA Marker; N: Blank; Lane 1: Hp standard strain; Lane 2: Escherichia coli; Lane 3: Staphylococcus aureus; Lane 4: Campylobacter rectus; Lane 5: Streptococcus salivarius; Lane 6: Lactobacillus.
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多重PCR灵敏性试验 Multiplex PCR sensitivity test. M: D2000 DNA Marker; N: Blank; Lane 1: 100 ng·μL-1; Lane 2: 10 ng·μL-1; Lane 3: 1 ng·μL-1; Lane 4: 1×10-1 ng·μL-1; Lane 5: 1×10-2 ng·μL-1; Lane 6: 1×10-3 ng·μL-1; Lane 7: 1×10-4 ng·μL-1.
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重组质粒PCR电泳结果 Recombinant plasmid PCR electrophoresis results. M: D2000 bp DNA Ladder; N1-N2: Blank; Lanes 1-3: pGMT-16s; Lanes 5-7: pGMT-cagA.
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多重PCR扩增临床唾液标本Hp16S rRNA及cagA基因凝胶电泳图谱 Gel electrophoresis profiles of multiplex PCR amplification of Hp 16S rRNA gene and cagA gene in clinical saliva samples. M: D2000 DNA Marker; N: Blank; Lane 1: pGMT-Hp; Lane 2: Saliva specimen No. 14; Lane 3: Saliva specimen No. 60; Lane 4: Saliva specimen No. 136; Lane 5: Saliva specimen No. 22; Lane 6: Saliva specimen No. 96; Lane 7: Saliva specimen No. 120; Lane: 8 Saliva specimen No. 7.
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