EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

U.S Environmental Protection Agency

U.S. Environmental Protection Agency

Using robotics and the MPN technique, a 96-microwell method was developed to compare two procedures for enumeration of viable chlorine-treated B. atrophaeus spores: broth-culture enrichment followed by real-time polymerase chain reaction analysis; and filter plating on agar. Recoveries of chlorine-treated spores were improved by broth enrichment over filter plating, whereas recoveries of non-treated spores were not different in the two procedures.

An integrated culture and real-time PCR method to assess viability of disinfectant treated Bacillus spores using robotics and the MPN quantification method.

&#x9690;&#x5B62;&#x5B50;&#x866B;&#x662F;&#x91CD;&#x8981;&#x7684;&#x6C34;&#x6027;&#x539F;&#x751F;&#x52A8;&#x7269;&#x5BC4;&#x751F;&#x866B;&#x53EF;&#x5BFC;&#x81F4;&#x4E25;&#x91CD;&#x8179;&#x6CFB;&#x548C;&#x6B7B;&#x4EA1;&#x4E2D;&#x514D;&#x75AB;&#x529B;&#x4F4E;&#x4E0B;&#x3002;&#x76EE;&#x524D;&#x7684;&#x65B9;&#x6CD5;&#x7528;&#x6765;&#x76D1;&#x89C6;&#x7684;&#x6C34;&#x4E2D;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x662F; 1622&#x5E74;&#x548C; 1623&#x5E74;&#x57FA;&#x4E8E;&#x663E;&#x5FAE;&#x955C;&#x7684;&#x7F8E;&#x56FD;&#x73AF;&#x4FDD;&#x5C40;&#x65B9;&#x6CD5;&#x3002;&#x8FD9;&#x4E9B;&#x65B9;&#x6CD5;&#x8BC4;&#x4F30;&#x6E90;&#x6C34;&#x57DF;&#xFF0C;&#x866B;&#x5375;&#x56CA;&#x7684;&#x603B;&#x6C34;&#x5E73;&#xFF0C;&#x4F46;&#x5E76;&#x6CA1;&#x6709;&#x786E;&#x5B9A;&#x5375;&#x56CA;&#x6D3B;&#x529B;&#x6216;&#x57FA;&#x56E0;&#x578B;&#x3002;&#x6700;&#x8FD1;&#xFF0C;&#x4E19;&#x952D; monoazide (PMA) &#x5DF2;&#x88AB;&#x7528;&#x4E8E;&#x4E0E;&#x5206;&#x5B50;&#x8BCA;&#x65AD;&#x5DE5;&#x5177;&#x4E00;&#x8D77;&#x786E;&#x5B9A;&#x7269;&#x79CD;&#x548C;&#x8BC4;&#x4F30;&#x53EF;&#x884C;&#x6027;&#x7684;&#x7EC6;&#x83CC;&#x3002;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x7684;&#x76EE;&#x6807;&#x662F;&#x53D1;&#x5C55;&#x7684;&#x9690;&#x5B62;&#x5B50;&#x866B; PMA-&#xFF08;CryptoPMA-PCR&#xFF09; pcr&#xFF0C;&#x5305;&#x62EC; PMA &#x6CBB;&#x7597; PCR &#x5206;&#x6790;&#x524D;&#x7684;&#xFF0C;&#x4EE5;&#x9632;&#x6B62;&#x4ECE;&#x6B7B;&#x866B;&#x5375;&#x7684; DNA &#x6269;&#x589E;&#x3002;&#x7ED3;&#x679C;&#x8868;&#x660E; PMA &#x7A7F;&#x900F;&#x5176; DNA &#x53EA;&#x6B7B;&#x866B;&#x5375;&#x548C;&#x5757;&#x6269;&#x589E;&#x3002;CryptoPMA pcr &#x8FD8;&#x5177;&#x4F53;&#x53EF;&#x4EE5;&#x68C0;&#x6D4B;&#x6D3B;&#x866B;&#x5375;&#x56CA;&#x5185;&#x7684;&#x6D3B;&#x7684;&#x548C;&#x6B7B;&#x7684;&#x866B;&#x5375;&#x7684;&#x6DF7;&#x5408;&#x4EBA;&#x53E3;&#x3002;&#x66F4;&#x91CD;&#x8981;&#x7684;&#x662F;&#xFF0C;&#x6D3B;&#x866B;&#x5375;&#x56CA;&#xFF0C;&#x4E0D;&#x662F;&#x6B7B;&#x7684;&#x866B;&#x5375;&#xFF0C;&#x53D1;&#x73B0;&#x5728;&#x539F;&#x6599;&#x5E9F;&#x7269;&#x6216;&#x8868;&#x9762;&#x6C34;&#x63BA;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x7684;&#x6837;&#x672C;&#x3002;&#x6B64;&#x6982;&#x5FF5;&#x9A8C;&#x8BC1;&#x7814;&#x7A76;&#x662F;&#x7B2C;&#x4E00;&#x6B21;&#x8BC1;&#x660E; PMA &#x7528;&#x4E8E;&#x9884;&#x805A;&#x5408;&#x9176;&#x94FE;&#x53CD;&#x5E94;&#x6CBB;&#x7597;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x3002;CryptoPMA pcr &#x662F;&#x6709;&#x5438;&#x5F15;&#x529B;&#x7684;&#x529E;&#x6CD5;&#xFF0C;&#x6765;&#x5177;&#x4F53;&#x68C0;&#x6D4B;&#x548C;&#x57FA;&#x56E0;&#x578B;&#x53EF;&#x884C;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x4E2D;&#x7684;&#x6C34;&#xFF0C;&#x8FD9;&#x662F;&#x5F88;&#x5173;&#x952E;&#x7684;&#x4EBA;&#x4F53;&#x5065;&#x5EB7;&#x98CE;&#x9669;&#x8BC4;&#x4F30;&#x3002;

&#x9690;&#x5B62;&#x5B50;&#x866B;&#x4E19;&#x952D; monoazide PCR&#xFF0C;&#x57FA;&#x56E0;&#x5206;&#x578B;&#x7684;&#x53EF;&#x884C;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x7684;&#x4E00;&#x79CD;&#x57FA;&#x4E8E;&#x5206;&#x5B50;&#x751F;&#x7269;&#x5B66;&#x7684;&#x6280;&#x672F;&#x3002;

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

Cryptosporidium est un parasite protozoaire importante voie d&#x26;#39;eau qui peuvent causer une diarrh&#xE9;e grave et la mort chez les sujets immunod&#xE9;prim&#xE9;s. Les m&#xE9;thodes actuelles utilis&#xE9;es pour surveiller les oocystes de Cryptosporidium dans l&#x26;#39;eau sont les m&#xE9;thodes bas&#xE9;es sur la microscopie EPA 1622 et 1623. Ces m&#xE9;thodes d&#x26;#39;&#xE9;valuer les niveaux totaux d&#x26;#39;oocystes dans les eaux de source, mais ne d&#xE9;terminent pas la viabilit&#xE9; des oocystes ou g&#xE9;notype. R&#xE9;cemment, propidium monoazoture (PMA) a &#xE9;t&#xE9; utilis&#xE9; en conjonction avec des outils de diagnostic mol&#xE9;culaire pour identifier les esp&#xE8;ces et d&#x26;#39;&#xE9;valuer la viabilit&#xE9; des bact&#xE9;ries. L&#x26;#39;objectif de cette &#xE9;tude &#xE9;tait le d&#xE9;veloppement d&#x26;#39;un Cryptosporidium PMA-PCR (CryptoPMA-PCR) qui comprend le traitement PMA avant l&#x26;#39;analyse par PCR, afin d&#x26;#39;&#xE9;viter l&#x26;#39;amplification de l&#x26;#39;ADN &#xE0; partir des oocystes morts. Les r&#xE9;sultats ont d&#xE9;montr&#xE9; que la PMA ne p&#xE9;n&#xE8;tre que les oocystes de morts et d&#x26;#39;amplification des blocs de leur ADN. Le dosage CryptoPMA-PCR peut &#xE9;galement d&#xE9;tecter sp&#xE9;cifiquement oocystes vivants au sein d&#x26;#39;une population mixte d&#x26;#39;oocystes vivants et morts. Plus important encore, les oocystes vivants, des oocystes pas morts, ont &#xE9;t&#xE9; d&#xE9;tect&#xE9;s dans des d&#xE9;chets crus ou les &#xE9;chantillons d&#x26;#39;eau de surface enrichis avec des oocystes de Cryptosporidium. Cette &#xE9;tude de preuve de concept est la premi&#xE8;re &#xE0; d&#xE9;montrer l&#x26;#39;utilisation des PMA pour la pr&#xE9;-PCR de traitement des oocystes de Cryptosporidium. Le dosage CryptoPMA-PCR est une approche int&#xE9;ressante pour d&#xE9;tecter sp&#xE9;cifiquement et les oocystes de Cryptosporidium g&#xE9;notype viables dans l&#x26;#39;eau, ce qui est essentiel pour l&#x26;#39;&#xE9;valuation des risques pour la sant&#xE9; humaine.

Cryptosporidium propidium monoazoture-PCR, une biologie mol&#xE9;culaire bas&#xE9;e sur la technique pour le g&#xE9;notypage des oocystes de Cryptosporidium viables

Cryptosporidium ist eine wichtige Wasserwege-Protozoen-Parasit, der heftigen Durchfall und Tod in der immungeschw&auml;chten verursachen kann. Die aktuellen Methoden zur &Uuml;berwachung der Cryptosporidium-Oozysten im Wasser sind die Mikroskopie-basierte USEPA-Methoden 1622 und 1623. Diese Methoden insgesamt Ebenen von Oozysten im Quellcode Gew&auml;sser zu beurteilen, aber bestimmen nicht Oozyste Lebensf&auml;higkeit oder Genotypus. Vor kurzem wurde Propidium Monoazide (PMA) verwendet in Verbindung mit Molekulare Diagnose-Tools Arten zu identifizieren und bewerten die Lebensf&auml;higkeit der Bakterien. Das Ziel dieser Studie war die Entwicklung einer Cryptosporidium PMA-PCR (CryptoPMA-PCR)-Assay, der PMA-Behandlung vor der PCR-Analyse umfasst, um die Verst&auml;rkung von DNA von Toten Oozysten verhindern. Die Ergebnisse zeigten, dass PMA nur Tote Oozysten und Bl&ouml;cke Verst&auml;rkung von DNA dringt. Der CryptoPMA-PCR-Assay kann auch speziell live Oozysten innerhalb einer gemischten Population von lebenden und Toten-Oozysten erkennen. Noch wichtiger ist, Leben Oozysten, nicht tot Oozysten, gefunden in roher Verschwendung oder Oberfl&auml;che Wasser Proben gespickt mit Cryptosporidium-Oozysten. Diese Proof-of-Concept-Studie ist die erste, die Verwendung von PMA f&uuml;r Pre-PCR Behandlung von Cryptosporidium-Oozysten demonstrieren. Der CryptoPMA-PCR-Assay ist ein attraktives Konzept, speziell zu erkennen und Genotyp lebensf&auml;higen Cryptosporidium-Oozysten im Wasser, das f&uuml;r menschliche Gesundheit-Risikobewertung von entscheidender Bedeutung ist.

Cryptosporidium Propidium Monoazide-PCR, ein Molekularbiologie-basierte Verfahren zur Genotypisierung von lebensf&auml;higen Cryptosporidium-Oozysten.

Cryptosporidium &egrave; un importante a base acquosa protozoo parassita che pu&ograve; provocare diarrea grave e morte nei pazienti immunocompromessi. Gli attuali metodi utilizzati per monitorare le oocisti di Cryptosporidium in acqua sono i metodi USEPA basati su microscopia 1622 e 1623. Questi metodi valutare livelli totali di oocisti nelle acque di fonte, ma non determinano vitalit&agrave; oocisti o genotipo. Recentemente, propidium monoazide (PMA) &egrave; stato usato in combinazione con strumenti di diagnostica molecolari per identificare la specie e valutare la vitalit&agrave; dei batteri. L&#39;obiettivo di questo studio &egrave; stato lo sviluppo di un dosaggio di Cryptosporidium PMA-PCR (CryptoPMA-PCR) che include il trattamento di PMA prima analisi PCR al fine di evitare l&#39;amplificazione del DNA da oocisti morte. I risultati hanno dimostrato che la PMA penetra solo morto oocisti e blocchi di amplificazione del loro DNA. Il dosaggio di CryptoPMA-PCR &egrave; in grado di rilevare anche specificamente oocisti live all&#39;interno di una popolazione mista di oocisti vivi e morte. Ancora pi&ugrave; importante, dal vivo oocisti, oocisti non morte, sono state rilevate in rifiuti crudo o superficie acqua campioni diluiti con le oocisti di Cryptosporidium. Questo studio di proof-of-concept &egrave; il primo a dimostrare l&#39;uso di PMA per il trattamento pre-PCR di oocisti di Cryptosporidium. Il dosaggio di CryptoPMA-PCR &egrave; un approccio interessante per rilevare in modo specifico e oocisti di Cryptosporidium vitali genotipo in acqua, che &egrave; fondamentale per la valutazione dei rischi di salute umana.

Cryptosporidium propidium monoazide-PCR, una tecnica di base di biologia molecolare per la genotipizzazione di oocisti di Cryptosporidium vitali.

&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x306F;&#x514D;&#x75AB;&#x4E0D;&#x5168;&#x3067;&#x91CD;&#x5EA6;&#x306E;&#x4E0B;&#x75E2;&#x3084;&#x6B7B;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x91CD;&#x8981;&#x306A;&#x6C34;&#x7CFB;&#x539F;&#x866B;&#x5BC4;&#x751F;&#x866B;&#x3067;&#x3042;&#x308B;&#x3002;&#x6C34;&#x306E;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x73FE;&#x5728;&#x306E;&#x65B9;&#x6CD5;&#x306F;&#x3001;&#x9855;&#x5FAE;&#x93E1;&#x30D9;&#x30FC;&#x30B9;&#x306E;USEPA&#x30E1;&#x30BD;&#x30C3;&#x30C9;1622&#x3068;1623&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x306F;&#x3001;&#x30BD;&#x30FC;&#x30B9;&#x6C34;&#x4E2D;&#x306E;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x5408;&#x8A08;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x304C;&#x3001;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x53EF;&#x80FD;&#x6027;&#x3084;&#x907A;&#x4F1D;&#x5B50;&#x578B;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x308F;&#x3051;&#x3067;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3002;&#x6700;&#x8FD1;&#x3067;&#x306F;&#x3001;&#x30D7;&#x30ED;&#x30D4;&#x30B8;&#x30A6;&#x30E0;monoazide&#xFF08;PMA&#xFF09;&#x306F;&#x3001;&#x7A2E;&#x3092;&#x8B58;&#x5225;&#x3057;&#x3001;&#x7D30;&#x83CC;&#x306E;&#x751F;&#x5B58;&#x7387;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5206;&#x5B50;&#x8A3A;&#x65AD;&#x30C4;&#x30FC;&#x30EB;&#x3068;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3066;&#x4F7F;&#x7528;&#x200B;&#x200B;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x306E;&#x76EE;&#x7684;&#x306F;&#x3001;&#x6B7B;&#x8005;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x304B;&#x3089;&#x306E;DNA&#x306E;&#x5897;&#x5E45;&#x3092;&#x9632;&#x6B62;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;PCR&#x89E3;&#x6790;&#x306E;&#x524D;&#x306B;PMA&#x51E6;&#x7406;&#x3092;&#x542B;&#x3080;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;PMA-PCR&#xFF08;CryptoPMA-PCR&#xFF09;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x958B;&#x767A;&#x3067;&#x3057;&#x305F;&#x3002;&#x7D50;&#x679C;&#x306F;&#x3001;PMA&#x3060;&#x3051;&#x6B7B;&#x3093;&#x3060;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3068;&#x305D;&#x306E;DNA&#x306E;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x306E;&#x5897;&#x5E45;&#x3092;&#x8CAB;&#x901A;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002; CryptoPMA-PCR&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x307E;&#x305F;&#x3001;&#x7279;&#x306B;&#x30E9;&#x30A4;&#x30D6;&#x3084;&#x6B7B;&#x3093;&#x3060;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x6DF7;&#x5408;&#x96C6;&#x56E3;&#x5185;&#x3067;&#x306E;&#x30E9;&#x30A4;&#x30D6;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x91CD;&#x8981;&#x306A;&#x306E;&#x306F;&#x3001;&#x30E9;&#x30A4;&#x30D6;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3001;&#x6B7B;&#x3093;&#x3067;&#x3044;&#x306A;&#x3044;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306F;&#x3001;&#x751F;&#x30B4;&#x30DF;&#x3084;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3067;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x306E;&#x5730;&#x8868;&#x6C34;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x3067;&#x691C;&#x51FA;&#x3055;&#x308C;&#x305F;&#x3002;&#x3053;&#x306E;&#x6982;&#x5FF5;&#x5B9F;&#x8A3C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x4E8B;&#x524D;-PCR&#x6CBB;&#x7642;&#x306E;&#x305F;&#x3081;&#x306E;PMA&#x306E;&#x4F7F;&#x7528;&#x65B9;&#x6CD5;&#x3092;&#x793A;&#x3059;&#x305F;&#x3081;&#x306B;&#x6700;&#x521D;&#x306E;&#x3082;&#x306E;&#x3067;&#x3059;&#x3002; CryptoPMA-PCR&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x3001;&#x7279;&#x306B;&#x30D2;&#x30C8;&#x306E;&#x5065;&#x5EB7;&#x30EA;&#x30B9;&#x30AF;&#x8A55;&#x4FA1;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x6C34;&#x306B;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x306E;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3092;&#x691C;&#x51FA;&#x3057;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x578B;&#x306B;&#x9B45;&#x529B;&#x7684;&#x306A;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3067;&#x3059;&#x3002;

&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30D7;&#x30ED;&#x30D4;&#x30B8;&#x30A6;&#x30E0;Monoazide-PCR&#x3001;&#x751F;&#x304D;&#x305F;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x306E;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x30B8;&#x30A7;&#x30CE;&#x30BF;&#x30A4;&#x30D4;&#x30F3;&#x30B0;&#x306E;&#x305F;&#x3081;&#x306E;&#x5206;&#x5B50;&#x751F;&#x7269;&#x5B66;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x6280;&#x8853;

&#xC6C0;&#xC744; immunocompromised&#xC5D0; &#xC2EC;&#xD55C; &#xC124;&#xC0AC; &#xBC0F; &#xC8FD;&#xC74C;&#xC744; &#xC77C;&#xC73C;&#xD0AC; &#xC218; &#xC788;&#xB294; &#xC911;&#xC694; &#xD55C; waterborne protozoan &#xAE30;&#xC0DD;&#xCDA9; &#xC774;&#xB2E4;. &#xC6C0; oocysts &#xBB3C;&#xC5D0; &#xB300; &#xD55C; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xD604;&#xC7AC; &#xBA54;&#xC11C;&#xB4DC;&#xB294; 1622 &#xBC0F; 1623 &#xD604;&#xBBF8;&#xACBD;-&#xAE30;&#xBC18; USEPA &#xBC29;&#xBC95;. &#xC774;&#xB7EC;&#xD55C; &#xBC29;&#xBC95;&#xC740; oocysts &#xC18C;&#xC2A4; &#xBC14;&#xB2E4;&#xC5D0;&#xC11C;&#xC758; &#xC804;&#xCCB4; &#xC218;&#xC900;&#xC744; &#xD3C9;&#xAC00; &#xD558;&#xC9C0;&#xB9CC; oocyst &#xC0DD;&#xC874; &#xB610;&#xB294; &#xC720;&#xC804;&#xC790; &#xD615;&#xC744; &#xACB0;&#xC815; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xCD5C;&#xADFC;, propidium monoazide (PMA) &#xC218; &#xC885; &#xC2DD;&#xBCC4; &#xD558; &#xACE0; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC758; &#xC0DD;&#xC874; &#xB2A5;&#xB825; &#xD3C9;&#xAC00; &#xBD84;&#xC790; &#xC9C4;&#xB2E8; &#xB3C4;&#xAD6C;&#xC640; &#xD568;&#xAED8;&#xC5D0;&#xC11C; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC758; &#xBAA9;&#xD45C;&#xB294; &#xBC29;&#xC9C0; &#xC8FD;&#xC740; oocysts&#xC5D0;&#xC11C; DNA &#xC99D;&#xD3ED; &#xD558;&#xAE30; &#xC704;&#xD558;&#xC5EC; PCR &#xBD84;&#xC11D; &#xC804;&#xC5D0; PMA &#xCE58;&#xB8CC; &#xD3EC;&#xD568; &#xC6C0;&#xC744; PMA PCR (CryptoPMA-PCR) &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xAC1C;&#xBC1C; &#xD588;&#xB2E4;. &#xACB0;&#xACFC; PMA&#xC758; DNA&#xB9CC; &#xC8FD;&#xC740; oocysts &#xBC0F; &#xBE14;&#xB85D; &#xC99D;&#xD3ED; &#xCE68;&#xD22C; &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;. CryptoPMA-PCR &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xB77C;&#xC774;&#xBE0C;&#xC640; &#xC8FD;&#xC740; oocysts&#xC758; &#xD63C;&#xD569; &#xB41C; &#xC778;&#xAD6C; &#xB0B4;&#xC758; &#xB77C;&#xC774;&#xBE0C; oocysts &#xAC10;&#xC9C0; &#xAD6C;&#xCCB4;&#xC801;&#xC73C;&#xB85C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB354; &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC740;, &#xB77C;&#xC774;&#xBE0C; oocysts, &#xC544;&#xB2C8; &#xC8FD;&#xC740; oocysts &#xC6D0;&#xC2DC; &#xD3D0;&#xAE30;&#xBB3C; &#xB610;&#xB294; &#xD45C;&#xBA74;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC; &#xB41C; &#xC0D8;&#xD50C; &#xC6C0; oocysts&#xB85C; &#xC544;&#xAD70;&#xC744; &#xBB3C;. &#xC774; &#xAC1C;&#xB150; &#xC99D;&#xBA85; &#xC5F0;&#xAD6C;&#xB294; &#xBA3C;&#xC800; &#xC6C0; oocysts&#xC758; pre-PCR &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; PMA&#xC758; &#xC0AC;&#xC6A9; &#xBC29;&#xBC95;&#xC744; &#xC124;&#xBA85; &#xD558;&#xAE30;. CryptoPMA-PCR &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xAD6C;&#xCCB4;&#xC801;&#xC73C;&#xB85C; &#xAC10;&#xC9C0; &#xD558;&#xB294; &#xB9E4;&#xB825;&#xC801;&#xC778; &#xC811;&#xADFC; &#xBC0F; &#xC720;&#xC804;&#xC790; &#xD615; &#xAC00;&#xB2A5;&#xD55C; &#xC6C0; oocysts &#xC778;&#xAC04;&#xC758; &#xAC74;&#xAC15; &#xC704;&#xD5D8; &#xD3C9;&#xAC00; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xBB3C;&#xC5D0;&#xC11C;.

&#xC6C0; propidium monoazide-PCR, &#xAC00;&#xB2A5;&#xD55C; &#xC6C0; oocysts &#xC720;&#xC804;&#xD615; &#xBD84;&#xC790; &#xC0DD;&#xBB3C;&#xD559; &#xAE30;&#xBC18; &#xAE30;&#xC220;.

Cryptosporidium es un protozoario par&#xE1;sito importantes transmitidas por el agua que puede causar diarrea severa y la muerte en los pacientes inmunodeprimidos. Los m&#xE9;todos actuales utilizados para detectar ooquistes de Cryptosporidium en el agua son los m&#xE9;todos basados &#x200B;&#x200B;en microscop&#xED;a USEPA 1622 y 1623. Estos m&#xE9;todos de evaluar los niveles totales de ooquistes en las fuentes de agua, pero no determinar la viabilidad de los ooquistes o genotipo. Recientemente, propidio monoazide (PMA) se ha utilizado en conjunci&#xF3;n con moleculares herramientas de diagn&#xF3;stico para identificar las especies y evaluar la viabilidad de las bacterias. El objetivo de este estudio fue el desarrollo de un Cryptosporidium PMA-PCR (CryptoPMA-PCR) que incluye el tratamiento de PMA antes del an&#xE1;lisis de PCR con el fin de evitar la amplificaci&#xF3;n de ADN de ooquistes muertos. Los resultados demostraron que el PMA s&#xF3;lo penetra ooquistes muertos y amplificaci&#xF3;n de los bloques de su ADN. El ensayo CryptoPMA-PCR tambi&#xE9;n se puede detectar ooquistes en concreto en vivo dentro de una poblaci&#xF3;n mixta de oocistos vivos y muertos. M&#xE1;s importante a&#xFA;n, los ooquistes en vivo, los ooquistes no muertos, se han detectado en los residuos de crudos o las muestras de aguas superficiales enriquecidas con ooquistes de Cryptosporidium. Este estudio de prueba de concepto es el primero en demostrar el uso de los PMA para el tratamiento pre-PCR de ooquistes de Cryptosporidium. El ensayo CryptoPMA-PCR es un enfoque atractivo para la detecci&#xF3;n espec&#xED;fica y ooquistes de Cryptosporidium genotipo viables en el agua, lo cual es cr&#xED;tico para la evaluaci&#xF3;n del riesgo para la salud humana.

Cryptosporidium propidio Monoazide-PCR, uno de Biolog&#xED;a Molecular basada en la t&#xE9;cnica de tipificaci&#xF3;n gen&#xE9;tica de las viables ooquistes de Cryptosporidium

The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

Development and evaluation of EPA method 1615 for detection of enterovirus and norovirus in water.

To better understand and study the infection of the protozoan parasite Cryptosporidium parvum, a more sensitive in vitro assay is required. In vivo, this parasite infects the epithelial cells of the microvilli layer in the small intestine. While cell infection models using colon, kidney, and stomach cells have been studied to understand the infectivity potential of the oocysts, an ideal in vitro model would be readily-available, human-derived, and originating from the small intestine. In this study, we developed a reproducible, quantitative infection model using a non-carcinoma, human small intestinal epithelial cell type, named FHs 74 Int. Our results show that FHs 74 Int cells are productively infected by viable oocysts, and exhibit higher levels of infection susceptibility compared to other cell types. Moreover, infection rate of the sporozoites on the monolayer was found to be comparable or better than other cell types. We furthermore demonstrate that infection can be improved by 65% when pre-treated oocysts are directly inoculated on cells, compared to inoculation of excysted sporozoites on cells. Identification of a better infection model, which captures the preferred site of infection in humans, will facilitate studies on the host pathogenesis mechanisms of this important parasitic human pathogen.

A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum.

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.

Eunice A. Varughese

National Exposure Research Laboratory

U.S Environmental Protection Agency

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

Eunice A. Varughese

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}National Exposure Research Laboratory

U.S Environmental Protection Agency

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

National Exposure Research Laboratory